8- Dr Mohebali Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 60 Original Article Neospora caninum and Leishmania infantum Co-Infection in Domestic Dogs (Canis familiaris) in Meshkin-Shahr District, Northwestern Iran M Sharifdini1, ∗M Mohebali1, H Keshavarz1, M Hosseininejad2, H Hajjaran1, B Akhoundi1, A Rahimi Foroushani3, Z Zarei1, S Charehdar1 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Veterinary Parasitology, Faculty of Veterinary Medicine and Research Institute of Zoonotic Diseases, University of Shahrekord, Shahrekord, Iran 3Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 26 Oct 2010; accepted 13 Jul 2011) Abstract Background: Mediterranean visceral leishmaniasis (MVL) is an infectious disease that affects both human and ani- mals. Domestic dogs (Canis familiaris) are principal reservoir hosts of MVL caused by Leishmania infantum. Dogs are definitive hosts for Neospora caninum and a risk factor for infecting intermediate hosts. The immunosuppression caused by visceral leishmaniasis (VL) can promote the occurrence of co-infections with other agents such as neosporosis. This study aimed to determine the frequency of co-infection of the both protozoan parasites in the en- demic areas of VL from Meshkin-Shahr District, north-west of Iran. Methods: Altogether, 171 serum samples were collected from domestic dogs of Meshkin- Shahr District by multistage cluster sampling from October 2008 to August 2009. The collected serum samples were tested for the detection of simultaneous infection of L. infantum and N. caninum using direct agglutination test (DAT) and indirect ELISA, respectively. Results: Of the 171 domestic dogs, 27 (15.8%) and 52 (30.4%) were showed antibodies against L. infantum and N. caninum, respectively. Simultaneous infections of N. caninum and L. infantum was found in 16 (9.4%) of the dogs. In VL-positive and VL-negative dogs, N. caninum infection was found in 59.3% and 25.0%, respectively. A statistically significant difference was found between VL-positive and VL-negative dogs with N. caninum infection (P= 0.001). Conclusion: These findings indicate that Meshkin-Shahr District in northwestern Iran is an active focus of canine visceral leishmaniasis (CVL). Neospora caninum and L. infantum co-infection is prevalent in the area and infection by L. infantum seems to enhance susceptibility to N. caninum infection in domestic dogs. Keywords: Neospora caninum, Leishmania infantum, Co-infection, Seroepidemiology, dog, Iran Introduction Canine visceral leishmaniasis (CVL) is an infectious disease that affects both human and canines, and is transmitted by phleboto- mine sand flies (Alvar et al. 2004). Leishma- nia infantum is the agent of Mediterranean visceral leishmaniasis and domestic dogs are efficient reservoir hosts for this infection (WHO 1990). Asymptomatic infected dogs can re- main infected without displaying apparent cli- nical manifestations of visceral leishmaniasis for years and even for their whole life (Moshfe et al. 2009). Many countries have re-emer- gence of visceral leishmaniasis (VL) during re- cent years, in both aspects of geographically *Corresponding author: Dr Mehdi Mohebali, E-mail: mohebali37@yahoo.com Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 61 and the number of cases (Arias et al. 1996). For a long time, kala-azar seems to be en- demic in Meshkin-Shahr areas from the north- west of Iran (Moshfe et al. 2008). Infected dogs are important risk factors for human in- fection in the endemic areas of Iran (Gavgani et al. 2002). The prevalence of L. infantum in- fection in the domestic dogs in Meshkin-Shahr areas is high and its prevalence has been re- ported from 14.2% to 17.4% (Bokaei et al. 1998, Mohebali et al. 2005, Moshfe et al. 2008). Neospora caninum is a coccidian intra- cellular parasite from the Sarcocystidae fam- ily with a wide host range (Dubey and Lind- say 1996). Dogs, coyotes, and dingoes are both the definitive and intermediate hosts for N. caninum that excreted oocysts in their fe- ces and shedding oocysts in the environment is a risk factor for the occurrence of miscar- riages and stillbirths associated with N. can- inum in cattle and probably other intermedi- ate hosts (Dubey 2003, King et al. 2010). The parasite can be transmitted transplacentally in dogs for several generations (Dubey and Lindsay 1989). Neosporosis can cause severe neuromuscular disorders as ascending paraly- sis with hyperextension of the hind limbs, especially in congenitally infected dogs (Dubey 2003). A few studies on N. caninum infec- tion in dogs have been carried out in Iran (Haddadzadeh et al. 2007, Malmasi et al. 2007, Hosseininejad et al. 2010a, 2010b, Yakhchali et al. 2010). Studies on N. caninum and L. infantum co-infection in the world are very rare (Andreotti et al. 2006, Cringoli et al. 2002). This is the first study in Iran, aimed to determine simultaneous infection of N. can- inum and L. infantum in domestic dogs from Meshkin-Shahr District as CVL endemic area in north-west of Iran. Materials and Methods Animals A total of 171 domestic dogs of mix breeds from some villages of Meshkin-Shahr areas in- cluding Ahmad abad, Paikhan, Kojanagh, Mazraeh khalaf, Mirak and Ourkandi were selected by multi-stage cluster sampling from October 2008 to August 2009. Information on the dogs such as sex and age were taken from the owners and physical examination was performed by a doctor of Veterinary Medicine (DVM). Preparation of the serum samples All blood samples were prepared from the cephalic vein by venapuncture, poured into 10 ml polypropylene tubes, and processed 4– 10 h after collection. The collected samples were centrifuged at 800 g for 5-10 min, and the separated sera were kept at -20° C until tested. Direct agglutination test Serum samples were tested using direct agglutination test (DAT) for the detection of anti-L. infantum antibodies.. DAT antigen was prepared at the School of Public Health, Tehran University of Medical Science (Mohe- bali et al. 2006). The main phases of the process of making DAT antigen were mass cultivation of pro- mastigotes of Iranian strain of L. infantum (MCAN/IR/96/Lon49) in RPMI1640 plus 10% fetal bovine serum, tripsinization of the para- sites, staining with Coomassie brilliant blue and fixing with 1.2% formaldehyde (Mohebali et al. 2006). To screen the serum samples, initially dilutions were made 1: 80 and 1: 320 and sam- ples with titers 1: 80 were diluted further to give the end-point dilution of 1: 20480. Negative control (antigen only) and known positive con- trol serum samples were included on each plate. The cut off titer was defined as the highest dilution at which agglutination was still visible, as blue dot, compared with negative control wells, which had clear blue dots (Mohebali et al. 2005). The positive control serum was produced from dogs with L. infantum infection (at 1: 20480) and confirmed by microscopy, culture and animal inoculation (Harith et al. Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 62 1989). Based on previously studies in Iran, Anti-Leishmania antibodies at a titer of 1: 320 and higher were considered as positive in this investigation (Mohebali et al. 2006). Indirect ELISA For the detection of N. caninum infec- tion, sera were tested with an indirect ELISA using an affinity purified surface antigen (P38). The performance of indirect ELISA was based on the method of Hosseininejad et al. (Hossein- inejad et al. 2010a). Optical density (OD) values were measured at 450nm on an ELISA reader (Labsystems Multiskan ® plus). An optimal serum dilution of 1:100 was established by checkerboard titration for the purpose of the test. Index values were calcu- lated by the formula; SIn= (Sn−N)/(P−N) where SIn is the individual ELISA index value, Sn is the OD value obtained for the mean of tested samples, N is the OD value obtained for the negative serum, and P repre- sents the OD values obtained for the positive serum. Anti- Neospora antibodies at OD≥ 0.23 by ELISA were considered as Neospora sp. positive results (Hosseininejad et al. 2010 a). Parasitological study To confirm visceral leishmaniasis in dogs, necropsy was performed in some of high sero- positive dogs with specific clinical signs and symptoms. Liver and spleen samples of the in- fected dogs cultured in specific media such as NNN and RPMI1640 and then a few touch smears were prepared from the liver and spleen of them. All the prepared smears were fixed with methanol, stained with Giemsa stain 10% and examined microscopically for the presence of amastigotes. Ethical approval This research project was reviewed and approved by the Ethics Committee of Tehran University of Medical Sciences, Iran. Statistical analysis Statistical test including Chi-squared (X2) and MacNemar were used to compare sero- prevalence values relative to gender, age and clinical signs of N. caninum and L. infantum co-infection in the domestic dogs. Statistical analyses were performed using SPSS software version 13.5 (SPSS Inc, Chicago, IL, USA), with a probability (P) value of <0.05 as statis- tically significant. Results All of the dogs were of mixed breed and all of them were kept as guard dogs and sheep-dogs. From 171 serum samples that were tested by DAT, 27 (15.8%) of them were positive in with DAT (≥1:320). The sero- prevalence values among male and female animals were 16.4% and 12.0%, respectively (Table 1). No statistically significant differ- ence was seen between canine L. infantum in- fection and the gender (P= 0.574). The highest sero-prevalence rate 9/36 (25.0%) was ob- served among the domestic dogs of more than five years age and the lowest values 3/52 (5.8%) were seen in dogs under 2 years old (Table 2). Clinical signs including dermatitis, alopecia, keratitis, cachexia and weakness were seen in 27/171(15.8%) dogs. Only 12 (44.4%) from 27 DAT- seropositive dogs (≥1:320) showed clinical signs (Table 3). In this survey, 8 out of 12 necropsied dogs were positive for L. infantum infection with DAT (≥1:320) and impression smear that prepared from their liver and spleen. Only 3 out of 12 necropsied dogs showed positive results in Leishmania culture media about 2 weeks post inoculation. Altogether, 52 (30.4%) of the dogs showed anti N. caninum antibodies by indi- rect ELISA test using an affinity purified surface antigen (P38). No significant statisti- cally difference was seen between N. can- inum infection and both sex and age groups (P> 0.05) (Table 1 and 2). None of the in- fected dogs had clinical signs including neu- rologic and cutaneous neosporosis disorders. Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 63 The presence of antibodies against N. caninum and to L. infantum was detected in 16 from 171 dogs (9.4%) among six villages of Meshkin-Shahr, which were selected by multi-stage cluster sampling (Fig.1). L. infantum infection seems to enhance susceptibility to N. caninum infection in do- mestic dogs. [κ2= 10.26, P< 0.01, odds ratio= .43 (95% CI, 0.25–0.75). Table 4 and 5 show the distribution of the N. caninum infection in VL-negative dogs and VL-positive dogs, respectively. In DAT- positive dogs (≥1:320), the prevalence of N. caninum infection was calculated 59.3% and in DAT-negative dogs (<1: 320) the preva- lence of the infection was 25.0%. A statisti- cally significant difference was found between DAT-positive and DAT-negative dogs com- pared with N. caninum infection (P= 0.001). In 58.3% of males and 66.7% of female in the DAT-positive dogs, showed anti N. can- inum antibodies (OD≥ 0.23). No statistically significant difference was found in the both groups (P= 0.781). Also, 25.4% of males and 22.7% of female in the DAT- negative dogs, showed anti N. caninum antibodies but no statistically significant difference was found between in the both groups (P= 0.789). Fig. 1. Frequency of Neospora caninum and Leishmania infantum co-infection among the domestic dogs were selected by multi-stage cluster sampling in Meshkin-Shahr District, northwestern Iran Table 1. Sero-prevalence of canine L. infantum and N. caninum co-infection by gender in Meshkin-Shahr District, northwestern Iran Gender n of dogs (%) Leishmania Positive* Neospora positive** n (%) n (%) Male 146 (85.4) 24 16.4 45 30.8 Female 25 (14.6) 3 12.0 7 28.0 Total 171 (100) 27 15.8 52 30.4 * anti-Leishmania antibodies detection by DAT at titers ≥1:320 **anti-Neospora antibodies detection by ELISA at OD≥0.23 No statistically significant difference between DAT positive dogs (≥1:320) and gender was observed (P= 0.574). No statistically significant differenc between N. caninum infection and gender was observed (P= 0.777). L. infantum infection could increase N. caninum infection in the domestic dogs [κ2=10.26, P<0.01, odds ratio=0.43 (95%CI, 0.25–0.75)] Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 64 Table 2. Sero-prevalence of canine L. infantum and N. caninum co-infections by age group in Meshkin-Shahr District, northwestern Iran Age group (years) n of dogs (%) Leishmania Positive* Neospora positive** n (%) n (%) < 2 52 (30.4) 3 5.8 12 23.1 2-5 83 (48.5) 15 18.1 29 34.9 > 5 36 (21.1) 9 25.0 11 30.6 Total 171(100) 27 15.8 52 30.4 * anti-Leishmania antibodies detection by DAT at titers ≥1:320 **anti-Neospora antibodies detection by ELISA at OD≥0.23 Statistical difference between DAT positive dogs (≥1:320) and different age groups is significant (P= 0.038). No statistically significant difference between N. caninum infection and different age groups was observed (P= 0.345). Table 3. Distribution of symptomatic and asymptomatic dogs by sero-prevalence rate of L. infantum infection in Meshkin-Shahr District, northwestern Iran DAT test (≥1:320) n of dogs (%) Symptomatic Asymptomatic _______________________________ n % n % Positive 27 (15.8) 12 44.4 15 55.6 Negative 144 (84.2) 15 10.4 129 89.6 Total 171 (100) 27 15.8 144 84.2 Statistical difference between DAT positive (≥1:320) symptomatic and asymptomatic dogs was significant (P=0.001). Table 4. Distribution of N. caninum infection by gender and age in DAT - dogs from Meshkin-Shahr District, northwestern Iran Variables N. caninum N. caninum Total Positive Negative n % n % Sex Males 31 25.4 91 74.6 122 Females 5 22.7 17 77.3 22 Total 36 25 108 75 144 Age < 2 10 20.4 39 79.6 49 2-5 21 30.9 47 69.1 68 > 5 5 18.5 22 81.5 27 Total 36 25 108 75 144 In DAT negative dogs, no statistically significant difference was found between N. caninum infection with gender (P= 0.789) and age groups (P= 0.299). Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 65 Table 5. Distribution of N. caninum infection by gender and age in DAT+ dogs from Meshkin-Shahr District, northwestern Iran Variables N. caninum N. caninum Total Positive Negative n % n % Sex Males 14 58.3 10 41.7 24 Females 2 66.7 1 33.3 3 Total 16 60 11 40 27 Age < 2 2 66.7 1 33.3 3 2-5 8 53.3 7 46.7 15 > 5 6 66.7 3 33.3 9 Total 16 60 11 40 27 In DAT positive dogs, no statistically significant difference was found between N. caninum infection with gender (P= 0.781) and age groups (P= 0.782). Discussion For diagnosing canine leishmaniasis, DAT is considered a sensitive and applicable technique and is well correlated with clinical signs (Moshfe et al. 2008). According to pre- vious studies (Harith et al. 1989, Edrissian et al. 1996, Mohebali et al. 2004) the perform- ance of the DAT for recognizing of VL in dogs was desirable. Hence, we utilized DAT for the determination of sero-prevalence of canine L. infantum infection in this inves- tigation. Based on the present study, sero-preva- lence of canine Leishmania infection in the domestic dogs of Meshkin-Shahr District was determined 15.8% using DAT with cut- off value of 1:320. Based on three studies that designed for sero-prevalence of canine leishmaniasis in northwest of Iran, sero-posi- tivity rates of the infection were found 21.6%, 18.2% and 17.4%, respectively (Gav- gani et al. 2002, Mohebali et al. 2005, Moshfe et al. 2008). In this study, 55.6% of the DAT- positive dogs did not show any clinical signs. In Iran, it found asymptomatic Leishmania-infected dogs as well as sympto- matic ones, have a potential role in the main- tenance of L. infantum infection and proba- bly the establishment of domestic transmis- sion cycle of the parasite in the VL endemic areas (Moshfe et al. 2009). The seroprevalence values among male and female animals were 16.4% and 12.0%, respectively No statistically significant dif- ference between canine Leishmania infection and gender was observed (P=0.083). Similar findings were reported by other Iranian authors (Bokaei et al. 1998, Mohe- bali et al. 2005, Moshfe et al. 2008). Our findings indicated that the chance of having L. infantum antibodies increases with age of the dogs is in agreement with previous stud- ies (Mohebali et al. 2005, Moshfe et al. 2008). Statistical analysis revealed greater sero-prevalence in older dogs (5 years and above), indicating that the chance of expo- sure to the bite of sand flies infected with L. infantum increases when the dogs become older (Cardoso et al. 2004). In the current study, the prevalence of anti -N. caninum antibodies was determined 30.4% in dogs of Meshkin-Shahr District. This sero-prevalence was reported to be 33%, 28% and 27% in Iran, respectively (Haddad- zadeh et al. 2007, Malmasi et al. 2007, Yakhchali et al. 2010). These higher sero- prevalences in dogs can be due to the close Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 66 contact with placenta, materials of aborted fetuses, and the uterine discharge of the intermediate hosts of the parasite as cattle (Dijkstra et al. 2002, Fernandes et al. 2004). No significant difference was found between N. caninum sero-positivity male dogs and females (P> 0.05). This finding has agreement with the results of other studies (Haddad- zadeh et al. 2007, Malmasi et al. 2007, Yakhchali et al. 2010). In the present study, significant difference was not detected among the age groups (P> 0.05) but in other surveys in Iran the sero-positivity rates increased with age, suggesting postnatal exposure to N. caninum by means of horizontal transmis- sion (Haddadzadeh et al. 2007, Yakhchali et al. 2010). For the first time in Iran, we found the co-presence of antibodies to N. caninum and to L. infantum in 9.4% of domestic dogs. Cringoli et al. (2002) reported that 46 (4.3%) of 1058 dogs had co-presence of antibodies to N. caninum and to L. infantum. Based on these findings N. caninum and L. infantum co-infection is prevalent in this area and sig- nificant difference was found when sero- positive and sero-negative dogs for L. infan- tum were compared with N. caninum sero- prevalence. Sero-epidemiological surveys that were done in dogs from the Campania region of southern Italy indicated that the co- presence of antibodies of N. caninum and L. infantum is prevalent in dogs and infection by one protozoan seems to enhance the sus- ceptibility to the other one (Cringoli et al. 1996, 2002). Andreotti et al. (2006) reported that N. caninum and L. infantum co-infection is common in dogs in Campo Grande area, Brazil but there was no significant difference when VL-positive and VL-negative dogs were compared with N. caninum sero-preva- lence and VL does not appear to enhance susceptibility to N. caninum infection. In ad- dition, Tarantino et al. (2001) have reported N. caninum and L. infantum simultaneous skin infection in a young dog in Italy. In our study L. infantum infection seems to enhance susceptibility to N. caninum in- fection in domestic dogs (odds ratio=0.43) because canine visceral leishmaniasis may lead to immune system suppression and T-cell me- diated immune response to parasite antigens can be damaged. CD21+ and CD4+ lympho- cytes decline in the peripheral blood of in- fected dogs with CVL. Immune system sup- pression caused by CVL can be increased dog’s susceptibility to N. caninum infection (Tarantino et al. 2001). In conclusion, N. caninum and L. infan- tum co-infection in domestic dogs is preva- lent in the CVL endemic areas of northwest- ern Iran and the infection by L. infantum seems to enhance susceptibility to N. can- inum infection. Acknowledgements This investigation was supported fi- nancially by a Grant (No: 88-01-27-8485) from Vice- Chancellor for Research, Tehran Uni- versity of Medical Sciences and also was sup- ported by National Institute of Health Re- search (NIHR) of Islamic Republic of Iran. The authors thankful to Dr Tayebali, Dr A Ganji, Mr I Moradi, Mr E Taghdisi from Meshkin- Shahr Health Center and also from Mr M Nasiri, Mrs S Molaei, Dr GhR Khanbaba and other our colleagues for laboratorial and field activities. The authors declare that they have no conflict of interest. References Alvar J, Canavate C, Molina R, Moreno J, Nieto J (2004) Canine leishmaniasis. Adv Parasitol. 57: 1–88. Andreotti R, Oliveira JM, Silva EA, Oshiro LM, Matos Mde F (2006) Occurrence of Neospora caninum in dogs and its correlation with visceral leishmaniasis in the urban area of Campo Grande, Mato Grosso do Sul, Brazil. Vet Parasitol. 135: 375-379. Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 67 Arias JR, Monteiro PS, Zicker F (1996) The re-emergence of visceral leishmaniasis in Brazil. Emerg Infect Diseases. 2: 145-146. Bokaei S, Mobedi I, Edrissian GhH, Nadim A (1998) Seroepidemiological study of canine visceral leishmaniasis in Meshkin Shahr, northwest of Iran. Arch Inst Razi. 48–49: 41–46. Cardoso L, Rodrigues M, Santos H, Schoone GJ, Carreta P, Varejao E, van Benthem B, Afonso MO, Alves-Pires C, Semiao- Santos SJ, Rodrigues J, Schallig HD (2004) Sero-epidemiological study of canine Leishmania spp. infection in the municipality of Alijo (Alto Douro, Portu- gal). Vet Parasitol. 121: 21–32. Cringoli G, Capuano F, Veneziano V, Romano L, Solimene R, Barber JS, Trees A J (1996) Prevalence of antibodies against Neospora caninum in dog sera. Paras- sitologia. 38: 283. Cringoli G, Rinaldi L, Capuano F, Baldi L, Veneziano V, Capelli G (2002) Se- rological survey of Neospora caninum and Leishmania infantum co-infection in dogs. Vet Parasitol. 106: 307–313. Dijkstra T, Barkema HW, Eysker M, Hes- selink JW, Wouda W (2002) Natural transmission routes of Neospora caninum between farm dogs and cattle. Vet Para- sitol. 105: 99–104. Dubey JP (2003) Review of Neospora can- inum and neosporosis in animals. Korean J Parasitol. 41: 1–16. Dubey JP, Lindsay DS (1989) Transplacen- tal Neospora caninum infection in dogs. Am J Vet Res. 50: 1578–1579. Dubey JP, Lindsay DS (1996) A review of Neospora caninum and neosporosis. Vet Parasitol. 67: 1–59. Edrissian GhH, Hajjaran H, Mohebali M, Soleimanzadeh G, Bokaei S (1996) Application and evaluation of direct ag- glutination test in ser-diagnosis of vis- ceral leishmaniasis in man and canine reservoirs in Iran. Iranian J Med Sci. 21: 119–124. Fernandes BC, Gennari SM, Souza SL, Carvalho JM, Oliveira WG, Cury MC (2004) Prevalence of anti-Neospora can- inum antibodies in dogs from urban, periurban and rural areas of the city of Uberlandia, Minas Gerais-Brazil. Vet Parasitol. 123: 33–40. Gavgani AS, Mohite H, Edrissian GH, Mohebali M, Davies CR (2002) Do- mestic dog ownership in Iran is a risk factor for human infection with Leishma- nia infantum. Am J Trop Med Hyg. 67: 511–515. Haddadzadeh HR, Sadrebazzaz A, Malmasi A, Talei Ardakani H, Khazraii Nia P, Sadreshirazi N (2007) Seroprevalence of Neospora caninum infection in dogs from rural and urban environments in Te- hran, Iran. Parasitol Res. 101: 1563-1565. Harith A, Reiter I, Knapen F, Korte P, Huigen E, Kolk RHG (1989) Application of a direct agglutination test for detec- tion of specific anti-leishmania antibod- ies in the canine reservoir. J Clin Mi- crobiol. 27: 2252–2257. Hosseininejad M, Hosseini F, Mosharraf M, Shahbaz S, Mahzounieh M, Schares G (2010a) Development of an indirect ELISA test using an affinity purified sur- face antigen (P38) for sero-diagnosis of canine Neospora caninum infection. Vet Parasitol. 171(3–4): 337–342. Hosseininejad M, Hosseini F, Mahzounieh M, Raisi Nafchi A, Mosharraf M (2010b) Seroprevalence of Neospora caninum infection in dogs in Chaharma- hal-va-Bakhtiari Province, Iran. Comp Clin Pathol. 19: 269–270. King JS, Slapeta J, Jenkins DJ, Al-Qassab SE, Ellis JT, Windsor PA (2010) Aus- tralian dingoes are definitive hosts of Neospora caninum. Int J Parasitol. 40(8): 945–950. Iran J Arthropod-Borne Dis, 2011, 1(3): 60–68 M Sharifdini et al.: Neospora caninum and …. 68 Malmasi A, Hosseininejad M, Haddadzadeh H, Badii A, Bahonar A (2007) Serologic study of anti-Neospora caninum anti- bodies in household dogs and dogs living in dairy and beef cattle farms in Tehran, Iran. Parasitol Res. 100: 1143–1145. Mohebali M, Edrissian GhH, Nadim A, Hajjaran H, Akhoundi B, Hooshmand B, Zarei Z, Arshi S, Mirsamadi N, Manouchehri-Naeini K, Mamishi S, Sanati AA, Moshfeh AA, Charehdar S, Fakhar M (2006) Application of Direct agglutination test (DAT) for the diagnosis and seroepidemiological stud- ies of visceral leishmaniasis in Iran. Ira- nian J Parasitol. 1: 15–25. Mohebali M, Hajjaran H, Hamzavi Y, Mobedi I, Arshi S, Zarei Z, Akhoundi B, Naeini KM, Avizeh R, Fakhar M (2005) Epidemiological aspects of canine visceral leishmaniosis in the Islamic Re- public of Iran. Vet Parasitol. 129: 243– 251. Mohebali M, Taran M, Zarei Z (2004) Rapid detection of Leishmania infantum infec- tion in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination. Vet Para- sitol. 121: 239–245. Moshfe A, Mohebali M, Edrissian G, Zarei Z, Akhoundi B, Kazemi B, Jamshidi S, Mahmoodi M (2009) Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantum infec- tion. Acta Trop. 112: 101–105. Moshfe A, Edrissian GhH, Zarei Z, Ak- houndi B, Kazemi B, Jamshidi Sh, Mahmoodi M (2008) Seroepidemi- ological Study on Canine Visceral Leishmaniasis in Meshkin-Shahr District, Ardabil Province, Northwest of Iran during 2006–2007. Iranian J Parasitol. 3: 1–10. Tarantino C, Rossi G, Kramer LH, Perrucci S, Cringoli G, Macchioni G (2001) Leishmania infantum and Neospora caninum simultaneous skin infection in a young dog in Italy. Vet Parasitol. 102: 77–83. World Health Organization (WHO), (1990). Control of the leishmaniases WHO Technical Report Series 793: 27. Yakhchali M, Javadi S, Morshedi A (2010) Prevalence of antibodies to Neospora caninum in stray dogs of Urmia, Iran. Parasitol Res. 106(6):1455–1458.