J Arthropod-Borne Dis, December 2015, 9(2): 184–194 M Ansari-Mood et al.: Seroprevalence and Risk … 184 Original Article Seroprevalence and Risk Factors of Ehrlichia canis Infection among Companion Dogs of Mashhad, North East of Iran, 2009–2010 *Maneli Ansari-Mood 1, Javad Khoshnegah 1, Mehrdad Mohri 1, Seyed Mehdi Rajaei 2 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran 2Department of Clinical Sciences, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran (Received 17 Nov 2013; accepted 12 April 2014) Abstract Background: The aims of this study were to determine the seroprevalence of canine ehrlichiosis and risk factors of this disease in companion dogs’ population of Mashhad, North East of Iran. Canine Monocytic Ehrlichiosis (CME) is a zoonotic disease transmitted by ticks, Rhipicephalus sanguineus, and caused by an obligate intracellular bacterium, Ehrlichia canis. Methods: During September 2009 until November 2010, 250 companion dogs from Mashhad, North-East of Iran, were examined for serum antibody detection against E. canis by means of immunofluorescence assay test (IFAT) and factors associated with a positive antibody response. Results: There was a very low prevalence of anti-E. canis antibodies (0.8%, 2/250) among studied dogs. The anti- body titers for two seropositive dogs were 1:80 and 1:160, respectively. One (0.4%) of seropositive dogs was in- fested with, R. sanguineus. In blood smears from one of infested dogs (0.4%), typical morulae of E. canis was ob- served in lymphocytes. The results confirm that the lowest occurrence of reactive dogs indoors probably related to low tick infestion. Conclusion: This is the first report that describes serological evidences of canine monocytic ehrlichiosis in North- East of Iran. Results suggested that E. canis infection in owned pet dogs from North of Khorasan was not endemic from 2009 to 2010. Additional molecular studies are necessary to confirm E. canis infection and to identify the lo- cal strains of the organism. Keywords: Ehrlichia canis, Indirect Immunofluorescence Assay, Prevalence, Dog, IFA Introduction Canine Ehrlichiosis, a tick borne dis- ease, was first recognized by Donatien and Lestoquar (1935) and has since been re- ported in dogs geographical widespread (Bretischwerdt 1995). At the end of 1960 an epidemic outbreak of the disease with high mortality has been reported in Ameri- can military dogs and south Asia. This se- vere form was initially given the name Ca- nine Tropical Pancytopenia (William 1981). Ehrlichia species are bacteria of the family Anaplasmataceae. Ehrlichia canis is a gram negative highly pleomorphic, obli- gate intracellular bacterium which is envel- oped with a rippled thin outer membrane (Marvomatis et al. 2006). It is considered to be the major causative agent of Canine Monocytic Ehrlichiosis (CME) in dogs (Huxsoll et al. 1969). Rhipicephalus sanguineus, a brown-dog tick, kennel tick or pan-tropical dog tick belonging to Ixodidae family is a ubiqui- tous tick responsible for transmitting E. canis, (Jeremy et al. 2013). It is a one-host tick that feeds on dogs in all three stages of life cycle. Ticks acquire E. canis by feeding *Corresponding author: Dr Maneli Ansari-Mood, E-mail: Manelli.ansari@hotmail.com http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 184–194 M Ansari-Mood et al.: Seroprevalence and Risk … 185 on infected dogs and transmit infection for at least 155 days afterward to other dogs (Groves et al. 1975, Breitschwerdt et al. 1995). They can also act as vector of im- portant pathogens of humans such as Coxiella burnetii, Rickettsia conorii, R. rickettsii and Bartonella henselae being of zoonotic concern (Wikswo et al. 2007, Dantus et al. 2008). This tick species is known to be a vector of E. canis, Babesia canis, B. gipsony, Hepatozoon canis, and Anaplasma platys in dog (Gal et al. 2007, Anonymous. 2012). Three clinical stages have been proposed for CME, acute, subclinical and chronic. The acute phase is characterized by fever, anorexia, lymphadenomegaly, epistaxis and petechia (Neer and Harrus 2006). During the subclinical phase dogs appear healthy and have the potential to remain persistent carrier (Waner et al. 1996). In chronic cases, infected dogs fail to mount an effective im- mune response. Bone marrow involvement leads to pancytopenia (Moriera et al. 2005). The disease can be diagnosed by the detection of E. canis morulae in monocyte in blood smears or serologically detection of specific antibodies by the use of IFA test, dot-ELISA and Western blot immuno- assay or by the detection of E. canis in tis- sue and blood by means of PCR (Matthewman et al. 1993, Futch and Corstvet. 1996, Mylonakis et al. 2003). IFA is considered the “Gold standard” serologi- cal diagnostic technique for E. canis (Ristic et al. 1972). The objectives of this study were to determine the seroprevalance of canine ehrlichiosis and risk factors of this disease in companion dogs’ population of Mashhad, North Khorasan of Iran. Materials and Methods The study was performed on total 250 owned pet dogs (119 females and 131 males) between September 2009 until November 2010 referred to Veterinary Teaching Hos- pital of Ferdowsi University of Mashhad for their annual vaccination, as well as with clinical illness. The following details were obtained for each dog: sex, breed, age, body tempera- ture, location of dog’s home, appetite sta- tus, examination of lymph node, CRT, in- festation by tick, epistaxis and reason for referred to the hospital. After physical ex- amination blood samples were taken in EDTA and non-anticoagulant tube. Blood with EDTA were examined for hematology and complete blood count. Sera were separated by centrifuge and stored at -20 ºC until assayed. For each case blood smear was prepared and stained with Giemsa and direct microscopic examination was performed to detect Morula on white blood cells especially on monocytes and lymphocytes. Hematocrite and white blood cell count were recorded for all dogs. Anti- E. canis antibodies were detected by Flu Ehrlichia immune fluorescence kit (Flu EHRLICHIA Canis, Megacor, Aus- tria) with following method: Sera were added to the slides after dilu- tion (1:40) in phosphate-Buffered Saline (PBS) PH 7.2. Positive and negative control sera were also tested. Slides were Placed in humid chamber and incubated for 30 mi- nute at 37 ºC after that, those were washed twice in PBS. Then we added one drop anti-Dog FITC (conjugate) to each slides and those were returned to humid chamber and incubated for 30 minute at 37 ºC. Incu- bation was performed in the dark place to protect photosensitive conjugate. After the- se steps, slides were washed as described before and were air-dried then 2–3 drops of mounting fluid were added to each slides and a cover slip was placed. The slides were analyzed at ×400 magnification with IFA microscopy and were compared each wall with negative and positive control. Each serum sample at titer 1:40 or more http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 184–194 M Ansari-Mood et al.: Seroprevalence and Risk … 186 was considered positive. A positive reac- tion appears as bright sharp regularly stained inclusion bodies in cytoplasm of in- fected cell. The size, appearance and den- sity of the inclusion were compared with positive control. Sera were positive at the 1:40 were prepared serial dilution 1:80 1:160 1:320 1:640 1:1360 and tested again with IFA. All data were collected and because of low seropositive cases for E. canis, statisti- cal analysis was not performed. Results Complete blood count showed 67 ane- mic (26.8%), 40 thrombocytopenic (16%) dogs. Furthermore, 101 dogs in study pop- ulation were diagnosed with abnormal leukogram findings including 20 leukope- nia (8%), 24 leukocytosis (9.6%), 24 neu- tropenia (9.6%), 33 neutrophilia (13.2%). 7 dogs (2.8%) showed anemia and thrombo- cytopenia synchronously. 1 dog (0.4%) had morulae (Fig. 1). In physical examination, 12 dogs (4.8%) were infested with tick. All ticks were R. sanguineus. 15 dogs (6%) had lymph node enlargement, 6 dogs (2.4%) had fever and 4 dogs (1.6%) had epistaxis (Table 1). Two (0.8%) of the 250 dogs have been examined were found to be seropositive by the IFA. Both of them were adult (above 1 year) and the number of platelets, leuko- cytes and neutrophils were normal. Morula was found in lymphocytes of one the sero- positive dog. This dog showed inappetance and depression, had large submandibular lymph node and infested with R. sanguineus on physical examination. Fig. 1. A morulae of Ehrlichia canis (arrowed) in a blood smear from one of seropositive dogs (Morulae in cytoplasm of lymphocyte) Table 1. Signalment and antibody titer in seropositive dogs Variable Dog number 171 Dog number 235 Reference Ranges Age Sex Breed Hematocrit Thrombocyte Total WBC count Total Neutrophil count Morulae Body Temperature Appetite status Lymph nodes Epistaxis IFA titer 13 years old Female German shepherd 37 2.5 8000 6500 - 38.5 Normal Normal - 1:80 9 years old Male Mixed Terrier 42 3.34 7300 5986 In lymphocyte 39 Inappetite Submandibular L.n enlarged - 1:160 - - - 37-55 % 1.6-4.3×106/µ l 6000-17000/µ l 3000-11500/µ l - - - - - - http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 184–194 M Ansari-Mood et al.: Seroprevalence and Risk … 187 Discussion This study is the first investigation of the seroprevalence of E. canis antibodies among dogs in North Khorasan of Iran. The results revealed low prevalence of E. canis (2 dogs, 0.8%). Prevalence of ehrlichiosis was also re- ported from other regions of Iran: Jafari et al. (1997) in Shiraz (South west of Iran) have examined 180 dogs. Seventeen dogs (9.44%) were found positive for the pres- ence of E. canis in their white blood cells. Akhtardanesh et al. (2009) used IFA and ICA to detect antibodies against E. canis in 123 apparently healthy dogs. The overall seroprevalence was 14.63 %. Seventeen (13.8%) dogs in IFA test and 13 dogs (10.6%) in ICA were seropositive for CME. In blood smears from three infected dogs (16.6%) morulae were observed in mono- cytes. Avize et al. (2010) have reported seroprevalence of CME in 198 companion dogs of different ages by means of IFA and ICA 9.6 % in Ahvaz (West of Iran). Morulae of E. canis were observed in mon- ocyte of four infected dogs (2.02%). Blood samples from 980 dogs (510 do- mestic dogs and 475 wild dogs) in West Azerbaijan and 820 dogs (520 domestic dogs and 300 wild dogs) in East Azerbaijan of Iran were obtained by Asri and others in 2001 and tested by IFA for diagnosis of Canine Ehrlichiosis. Sixty seven percent of wild dogs and 38 % of domestic dogs in West Azerbaijan and for East Azerbaijan 58 % and 39 % were serologically positive for Ehrlichia. The main variants have been diagnosed were E. canis (75%), E. platys (20%) and E. equi (5%). In our study because of low seroprevalence of E. canis, we could not reach any correla- tion between age and CME but in many investigations the prevalence has been sig- nificantly differed among age groups. In Shiraz (Jafari et al. 1997) the animals of all ages seemed equally susceptible to disease. In Ahvaz (Avize et al. 2007) prev- alence rate have been significantly higher in adult dogs than juniors. The prevalence rate was 16.8 % in above 3 years old and 11.86 % in 1–3 years old compared with dogs less than 1 year old (1.41%). In Ker- man (Akhtardanesh et al. 2009) high asso- ciation was observed between age and se- ropositive dogs. Possible explanations for more infection in older group include the immunologic status of the host or more ex- posure to the vector ticks (Rodriguez-Vivas RI et al. 2005). German shepherd dog has been reported to be more susceptible to CME (Nyindo et al. 1980, Harrus et al. 1997). In Shiraz (Jafari et al. 1997) 21.1 % of infected dogs were German shepherd. Some research showed higher prevalence in male dogs (Batmaz et al. 2001, Costa et al. 2007). In some studies no significant difference was proved between sex and various breeds with presence of E. canis antibodies (Waner et al. 2000a, Rodriguez-Vivas et al. 2005, Hernandez et al. 2005, Solano- Gallego et al. 2006, Akhtardanesh et al. 2009, Avize et al. 2009, Roqueplo C et al. 2009). The clinical signs of CME may vary among and within geographic locations (Harrus et al. 1997a,b). The probable rea- sons include E. canis strain pathogenicity, dose of infectious organism, breed of dog, immuno status of the host and co-infection with other tick-borne parasites (Rodriguez- Vivas et al. 2005, Neer and Harrus 2006). Thrombocytopenia is the most common hematological finding in patients with acute CME. This change is found in all stage of disease but is more severe in chronic phase as a result of bone marrow hypoplasia (Troy et al. 1980). Death may occur as a http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 184–194 M Ansari-Mood et al.: Seroprevalence and Risk … 188 consequence of hemorrhages and secondary infections (Hendricks and Bob 2004). In our study because of low prevalence of E. canis we could not show any relationship between seropositive dogs and hemathologic changes but platelet and leukocyte count in both seropositive dogs were normal. In Ah- vaz (Avize et al. 2009) the prevalence of ehrlichiosis was higher in dogs with throm- bocytopenic although the difference was not significant and correlation was not ob- served between seronegative and seropositive dogs for hemathologic changes. In Kerman (Akhtardanesh et al. 2009) thrombocytope- nia, leukopenia and anemia were just ob- served in dogs with high IFA titer (>1:320). Rodriguez-Vivas RI et al. (2005) have found that the presence of thrombocytope- nia, platelet-related bleeding and a sero- positive response to E. canis in a patient in- crease the index of possibility for infection. The only known vectors of Ehrlichiae are ixodid ticks (Rikishia 1991). Rhipicephalus sanguineus and possibly the American dog tick, Dermacentor variabilis are the vector for E. canis (Groves et al. 1975, Johnson et al. 1998). Rhipicephalus sanguineus is widely distributed in the world but it is mainly in tropical and sub- tropical regions and also well adapted to the indoor environment where owned dogs are kept (Uspensku and Ioffe-Uspensky 2002, Dantas-Torres 2010). Dogs may ac- quire ticks in the city areas in parks and housing estates (Siuda 1993). Infestation by R. sanguineus has significant risk factor for E. canis seropositivity in Brazil (Trapp et al. 2002). In this study, 4.8 % (12 dogs) were infested by R. sanguineus. One of the seropositive dogs also had this tick on his trunk. R. sanguineus was also the most common species in North-East of Iran (Razmi et al. 2003). Diagnostic method can affect on preva- lence results of E. canis. As said above the indirect immunofluorescence antibody (IFA) test is considered the serological “gold standard” for diagnosis of CME (Ristic et al. 1972). Serological cross-reactivity occurs with other members of Ehrlichiae like E. equi (Baneth G et al. 1996), E. ristici (Ristici et al. 1999), E. ewingii (Anderson et al. 1992), E. chaffeensis, Neorickettsia helminthoeca (Rikisha 1991). In this study, IFA test was used and se- ropositive titers were 1:80 and 1:160. IFA test is more susceptible than other test but supplementary test such as PCR and west- ern immunoblotting is needed for detection of active infection and distinguished be- tween infections with different type of spe- cies. Possible explanations for low seropre- valence of E. canis in this study are: 1. Selected population: exposures to tick in domestic dogs are lower because of loca- tion and observance of health condition by owners. The life conditions of dogs affected the seroprevalence of E. canis (Roqueplo et al. 2009). Lim et al. (2010) indicate that risk of exposure to vector-borne disease in rural hunting dogs can be quite high in Korea. Ploneczka et al. (2003) showed that dogs in non-urban areas (9.9%) or they have living in outdoor (12.7%) had a higher prevalence of E. canis. Rural dogs had more parasite infestation than urban dogs (Dagnone et al. 2002, Carvalho et al. 2008). 2. Weather conditions: The prevalence of E. canis is largely dependent on the dis- tribution of the vector, R. sanguineus, which occurs mainly in tropical and sub- tropical regions but it has worldwide distri- bution (Jeremy et al. 2013). Jafari et al. (2008) have determined the prevalence of canine ectoparasite infesta- tion in pet dogs from the Shiraz area of southern Iran. Overall, 160 dogs were ex- amined for ectoparasites, and 142 R. sanguineus ticks were found on 13 dogs. A http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 184–194 M Ansari-Mood et al.: Seroprevalence and Risk … 189 significant correlation was observed between increases in temperature and decreases in humidity and increased ectoparasite infesta- tion. The number of dogs infested with ectoparasites in summer and spring was significantly higher than in winter (P= 0.007). Morales-Soto and Cruz-Vazquez (1998) found R. sanguineus along the year in Cuer- navaca, Mexico but the peaks of tick were found in April, July and November and the lower prevalence were in January. So season of sampling can affect seroprevalance of E. canis. 3. Type of serological test: IFA detects antibodies as early as 7 days after initial in- fection but some dogs may be negative un- til 28 days after infection or in acute phase of disease and also in chronic phase because of injury to immune system (Groves et al. 1975) when E. canis antibody titers results are negative, a follow up examination in 2 to 3 weeks or serotesting for other agents is recommended (Neer and Harrus 2006). The CME has a worldwide distribution and a significant seroprevalence in dogs from southeast Asia, Africa, Europe, Cen- tral and South America was reported (Car- denas et al. 2007). Antibodies against E. canis were detected in neighbors and close countries to Iran as 44.4 % in Saudi Arabia (Sacchini et al. 2007), 21 % in Turkey (Batmaz et al. 2001), and 33 % in Egypt (Botros et al. 1995). In our study, seroprevalence of E. canis was estimated less than 1 %. So CME is not endemic in Mashhad, but in Kerman, Ah- vaz and Azerbaijan is considered endemic (Asri et al. 2001, Akhtardanesh et al. 2009, Avizeh et al. 2010). Besides, E. canis is a human health haz- ard and causes clinical signs of disease (Pe- rez et al. 2006). Human Ehrlichiosis is caused by E. chaffeensis, A. phagocytophilum and E. ewingii (Dumler et al. 2007). Co-infec- tion of E. canis and A. phagocytophilum is possible (Amusategui et al. 2007). A. phagocytophilum was reported in Ixodes ricinus in North of Iran (Bashirbod et al. 2004). It is possible that more tick-transmitted pathogens can infect dogs, including E. canis, A. phagocytophilum, B. canis, Hepatozoon canis, Bartonella spp. (Baneth et al. 1998, Breitschwerdt et al. 1998, Yabsley et al. 2008). So in dogs with clinical signs of thrombocytopenia, leukopenia, fever and epistaxis if they have negative result for E. canis, consider the possibility of infectious with other organisms. There is significant correlation between ehrlichiosis with leptospirosis, leishmaniasis and babesiosis. So in dogs that have one of these diseases, E. canis infectious should be considered (Matthewman et al. 1993, Suksawat et al. 2001, Hernandez et al. 2005, Roura X et al. 2005, Tabar et al. 2009). Conclusion Ehrlichia canis infection in owned pet dogs from North of Khorasan was not en- demic from 2009 to 2010. Acknowledgements All financial support was provided by Ferdowsi University of Mashhad. Parts of sample processing and data analysis of this work are performed in the central labora- tory of Science and Research Branch, Is- lamic Azad University, Tehran, Iran with the assistance of Dr Seyed Mehdi Rajaei. The authors declare that there is no conflict of interests. 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