INTRODUCTION Canine ehrlichiosis has been observed as an emerging and earmarked rickettesial diseases, caused by rickettsial microorganism, Ehrlichia canis. It is currently reported throughout the world but at higher frequencies in tropical and subtropical regions due to presence of their vectors (Unver et al., 2003). The disease affects dogs, other domestic and wild animal species as well as humans (Perez et al., 2006). Rhipicephalus sanguineus, the brown dog tick, is the most widespread tick in the world and is a well-recognized vector of Ehrlichia canis and occasionally humans. The prevalence of E. canis is dependent on the distribution of the tick, which occurs mainly in tropical and subtropical regions. Ehrlichia canis is transmitted transtadially and intrastadially by Rhipicephalus sanguineus ticks (Bremer et al., 2005). It is a multisystemic disease with clinical symptoms like fever, leukopenia, thrombocytopenia, depression, anorexia, diarrhoea, depression, lethargy, neurological, ocular signs and bleeding through natural orifices in the form of epistaxis, hematemesis, hematuria and melena have been reported (Kumar and Varshney, 2006) which vary considerably in severity and frequency of occurrence in the initial and terminal phases of infection. The study has been designed with the objective of estimation of Canine ehrlichiosis in and around Rewa (M.P.). MATERIAL AND METHODS 200 dogs irrespective of age, sex and breed, with a history of anorexia and showing clinical signs of pale mucous membranes and dermal petechiae and with a history of recent tick infestation were screened for presence ehrlichia organism by blood smear examination and PCR assay. Thin blood smears were prepared after puncturing the ear tips with 24-gauge needle, fixed in methanol and stained with Giemsa and Leishman’s stains following standard procedures (Benjamin, 1982), which were observed under microscope for presence of ehrlichia organism and identified on the basis of characteristic morphology (Soulsby 1982). One ml blood samples were also obtained from suspected dogs from cephalic/saphenous vein for isolation of DNA for PCR and those showing band of 412 bpon PCR assay were included in the study. Molecular detection of Ehrlichia canis by PCR: Genomic DNA extraction: The blood samples collected in vaccutainer containing sodium EDTA were brought at room temperature and genomic DNA was extracted as follows: 1. Four hundred microliters of the sample was taken and centrifuged at 4,000 rpm for 3 min. 2. The cell pellets were resuspended in 1 ml of erythrocyte lysis solution, mixed, and centrifuged as described below: Erythrocyte lysis solution: 155 mM NH4Cl, M NaHCO3, disodium EDTA [pH 7.4] 3. Treatment with erythrocyte lysis solution was repeated until the leukocyte pellets lost all reddish colour.Template DNA was obtained as follows: (a) Four hundred microliters of lysis solution (2% Triton X-100, 1% sodium dodecyl sulfate, 100 mMNaCl, 10 mMTris-HCl [pH 8.0]) and 10 µl of proteinase K (10 mg/ml) were added to the samples, and the contents were mixed thoroughly and incubated for 30 min at 50ºC. (b) Four hundred microliters of saturated phenol was added, and the contents were mixed thoroughly and centrifuged at 10,000 rpmfor 10 min. (c) The aqueous layer was transferred to a fresh eppendorf tube, and an equal volume of chloroform-isoamyl alcohol (24:1) was added; the contents of tubes were mixed thoroughly and centrifuged at 10,000 rpmfor 10 min. (d) The upper layer was again transferred to a fresh eppendorf tube, and 200 µl of 7.5 M ammonium acetate was added and mixed thoroughly. Two volumes of absolute ethanol were added, the contents were mixed, and the tubes were stored at -20oC. (e) DNA was recovered by centrifuging the samples at 10,000 rpmfor 10 min. (f) The pellets were rinsed with 1 ml of 70% ethanol, dried, and resuspended in 30 µl of TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM disodium EDTA] and stored at -20oC until they were processed. Polymerase Chain Reaction: The PCR reaction was performed in 25 µl reaction volume containing 12.5 µl of 2X GoTaq® Green master mix (Promega, USA) (reaction buffer (pH 8.5), 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP and 3mM MgCl2) (Table 1), 1 µl of each forward and reverse primer (10 p-mole of each) (Integrated DNA Technology), 5 ul of DNA template and 5.5 µl of nucleic acid free distilled water to maintain the volume. The reaction was performed in thermal cycler (Applied Biosystem VeritiTM, ThermoFisher) with the lid temperature of 105oC with cycling condition as follows: • Initial denaturation at 96oC followed by 35 cycles of denaturation at 96oC for 45 sec, annealing at 59oC for 45 sec, extension at 72oC for 45 sec and final extension at 72oC for 7 min. • The amplified PCR products of 412 bp were subjected to electrophoresis in 1.5 % agarose gel (Bengaluru,Genei, India) with ethidium bromide at final concentration of 0.5µg/L and visualised in Gel documentation system (E-Gel Imager, Life Technologies, Invitrogen, USA) along with molecular marker (100 bp DNA ladder, Thermofisher, USA). RESULTS AND DISCUSSION Overall Prevalence: The overall prevalence of canine ehrlichiosis is shown in the Table 2. Out of the 200 dogs surveyed, 39 dogs were found positive showing an overall prevalence of canine ehrlichiosis as 19.5%. Our findings in the present study are in agreement with those of Katyal (2000); Dagnone et al. (2003) who reported a prevalence of 17.58% and 21.70% respectively. However, Bhadesiya and Raval (2015) reported the overall incidence of ehrlichiosis as 62.07% and Guedes (2015) reported an overall prevalence of 46.9%. Prevalence on the basis of blood smear examination and PCR Assay: Thin blood smears stained with Geimsa’s stain and PCR assay were used for the diagnosis of the canine ehrlichiosis. The results are shown in Table 3. Conventional microscopic parasitological diagnosis of the stained blood smear is the most commonly used techniques for the canine monocytic ehrlichiosis. But this is a challenging task, as sometimes the organism may not be detectedon peripheral thin blood smear, particularly seen in case of low parasitaemia or in chronic or inapparent infections. PCR based assays are utilized for the early diagnosis of the disease, especially in epidemiological surveys, owing to their higher sensitivity and specificity. In the present study, out of 200 dogs screened for ehrlichiosis, 14 dogs were found positive by blood smear method (7%) and 25 found positive by PCR method (12.5%). Higher prevalence of canine ehrlichiosis as detected by PCR based assays as compared to microscopy indicates the higher sensitivity levels of PCR. These findings are in agreement with the observations of Mittal et al. (2007); Parmar et al. (2013) Area-wise - Prevalence of E. canis. In the present study, a higher prevalence was recorded in rural area (19.81%) in comparison with urban area (19.14%) (Table 4). Higher prevalence in rural area may be due to unhygienic conditions and more tick population in these areas. Age-wise prevalence of E. canis. In the present study, maximum prevalence of canine ehrlichiosis was recorded in 1-3 years age group (30.76%), followed by less than 1 year age group (23.07%), 3-6 year age group (20.51%), 6-9 year age group (17.94%) and least prevalence was found in dogs more than 9 year age group (7.69%) (Table 5). Our findings corroborate with those of Karthika et al. (2014); Choudhary et al. (2012), who reported highest prevalence in dogs in the age group of 1-3 years. However, Rahman et al. (2010) reported no significant difference between age groups. Higher prevalence of canine ehrlichiosis was found in the age group of 1-3 years (30.76%) and lowest in dogs of more than 9 years. The reason for this is that the organism parasitizes monocytes. High bone marrow activity with active precursor cells i.e., monoblasts in young age gives an opportunity for the organism to parasitize more cells and multiply rapidly. The immune system in young animals is also in the developmental stage. However, many authors reported E. canis in different age group of dogs. Therefore, it can be opined that the age is not the criteria for Ehrlichia infection and it depends largely on the transmitting vector and the immune status of the host. Breed-wise prevalence of E. canis. The present study reported maximum prevalence in German Shepherd dogs (35.89%) followed by 30.76% in Non-Descript, 17.39% in Labrador, 7.69% in Pomeranian, 5.64% in Saint Bernard, and 2.56 % in Rottweiler (Table 6). These findings are in agreement with the earlier observations of Guedes et al. (2015); Singh et al. (2014), who reported higher prevalence in GSD. However, Tresamol et al. (1998) reported higher occurrence of canine ehrlichiosis in Doberman pinschers while and Costa et al., (2007) found it to be more prevalent in non-descript dogs. Higher susceptibility of GSD to the disease may be due to unnoticed tick infestation as the hair coat is comparatively thick with long hair. Sex-wise prevalence of E. canis. In the present study, higher prevalence was recorded in males (53.84%) as compared to females (48.71%) (Table 7). Similar findings were reported by Choudhary et al. (2012) and Kitaa et al. (2014). However, Tanikawa et al. (2013) reported no considerable differences between male and female dogs for E. canis infection. No sex wise predilection for the disease seems to be present as, it depends purely on the preference of the owners for keeping pets and generally males are more preferred over females as pets. Frequency of predominant signs of Canine Ehrlichiosis. Among the 18 positive dogs with canine ehrlichiosis, the characteristic clinical features observed were pyrexia (83.33%), lymphadenopathy (77.7%). anorexia (66.6%), depression and lethargy (66.6%), presence of ticks (61.1%), pale mucous membrane (55.5%), congested mucous membrane 26 (44.4%), melena (11.1%), epistaxis (33.3%), petechial hemorrhages (44.4%) (Table 8). These findings are in agreement with the earlier reports of Choudhary et al. (2015) and Sosa-Gutierrez et al. (2013). The clinical manifestations of ehrlichiosis in dogs varies between and within different geographical locations due to many factors like strain variations, breed of dogs infected, immunological status of the dog, concurrent infection with other tick borne infections. Elevated temperature, tachycardia and polypnoea were also reported by Choudhary et al. (2015). The clinical signs observed may be the result of inflammation which causes marked release of cytokine resulting in pyrexia and associated symptoms. The palpable lymph nodes were found to be enlarged in most of the dogs under study. Similar findings were recorded by Moreira et al. (2003). Intracytoplasmic leucocytic invasion by the rickettsia and due to generalized infection, lymphadenitis is observed. Replication of the organisms in the reticulo-endothelial system along with proliferation of medullary and para-cortical lymphocytes and aggregation of reactive histiocytes in the lymph nodes results in generalized lymphadenopathy. Pallor mucosae (55.5%) as observed in the present investigation in canine monocytic ehrlichiosis has been attributed to the loss of blood due to thrombocytopaenia, suppression of bone marrow and probably due to immune mediated red cell destruction. Whereas, bleeding tendencies (epistaxis, malena, haematemesis, petechial haemorrhages on oral gums and ventral abdomen) is mainly due to thrombocytopaenia and damage to vascular endothelium due to deposition of immune complexes on the vascular wall. Severe antibody mediated cytotoxic destruction of erythrocytes, release of pro-inflammatory cytokines in response to canine ehrlichiosis inhibiting the secretion of erythropoietin results in decreased RBC production together with immune mediated RBC destruction which are responsible for low RBC count and pale mucous membrane.