Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Molecular and Epidemiological Investigation with Haematobiochemical Profiling of Ehrlichia canis Infection in Dogs from Haryana, India

J
Jai Bhagwan1
0000-0002-9509-4067
Y
Yudhbir Singh1
0000-0001-5245-4731
R
Ricky Jhambh1
0000-0001-8039-8140
P
Parveen Kumar2,*
0000-0001-9627-3748
R
Ritik Kaul1
M
Mahavir Chaudhary3
D
Divya Agnihotri1
0000-0002-0719-9281
1Department of Veterinary Medicine, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.
2Department of Veterinary Microbiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.
3Department of Animal Genetics and Breeding, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.

ABSTRACT

Background: The study aimed to perform the molecular investigation, clinical observations and haemato-biochemical abnormalities associated with dogs naturally affected with canine monocytic ehrlichiosis (CME) from Haryana, India.

Methods: The study included 120 dogs suspected of haemoparasitic infection. History and clinical profiles were noted for each dog. PCR tested for four haemoparasites: H. canis, E. canis, B. vogeli and B. gibsoni. Seventeen dogs positive for E. canis underwent clinical investigation, haematological and biochemical analyses. Two 16s rRNA PCR products were sequenced and phylogenetic trees were prepared using MEGA X software. Data were analysed using SPSS 23.1. Parameters in CME-detected animals were compared with those in healthy animals.

Result: Most common clinical manifestations associated with CME were pyrexia, anorexia, a congested to pale mucous membrane and lethargy. Epistaxis, lymphadenomegaly, hindlimb weakness, melena and ocular discharge were among the less frequent clinical signs. Haematological findings revealed microcytic hypochromic to normochromic anaemia, thrombocytopenia and normal to raised leucocyte count with absolute neutrophilia and normal to raised lymphocyte count as the main observation. In affected dogs, serum biochemical profiles revealed normal to low protein levels with hypoalbuminemia, reduced A:G ratio, higher GGT activity and an elevated creatinine level. Phylogenetic analysis revealed that the partial 16s rRNA sequences of E. canis in the current study were similar to those of E. canis isolates from various regions of India and the world. The current study emphassis the significance of clinical, haematological and biochemical characteristics in the detection and treatment of dogs with CME.

INTRODUCTION

Ehrlichia belongs to the family Ehrlichiaceae, within the order Rickettsiales and the class Alphaproteobacteria. E. canis is commonly reported in India and causes classical canine monocytic ehrlichiosis (CME) (Megat Abd Rani et al., 2010). CME is a multisystemic disease, where acute form is characterised by lethargy, high fever, anorexia, lymphadenopathy, splenomegaly, epistaxis, dermal petechiae, ecchymoses and occasional tick infestation (Harrus and Waner, 2011). The chronic form presents with ulcerative stomatitis, necrotic glossitis, hind limb, scrotal oedema, pyoderma, icterus and central nervous system signs (Mylonakis and Theodorou, 2017). Microscopic detection of E. canis morula is a traditional and commonly used method (Mylonakis et al., 2003), but it lacks sensitivity during chronic and subclinical infections (Woody and Hoskins, 1991). Other common methods include serological assays like ELISA and IFA; however, these have low specificity and cannot differentiate between ongoing and past infections (Waner at al., 2001). Previous studies found that haemato-biochemical and clinical findings could aid early detection of E. canis in dogs (Bai et al., 2017; Sarma et al., 2015). Therefore, the current study aimed to investigate clinical and haemato-biochemical indicators for E. canis diagnosis in dogs. Previous studies have used PCR methods focusing on the 16s rRNA, p30 outer membrane protein, dsb and gp 19 gene regions (Stich et al., 2002; Nambooppha et al., 2018). In this study, we used the 16s rRNA region for PCR detection and genetic characterization of the organism.

MATERIALS AND METHODS

The Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS) Institutional Animal Ethics Committee approved the study’s sample collection and animal handling protocols on 25/03/2021 (VCC/IAEC/630-651). The present study was conducted on 120 dogs suspected to have haemoparasitic infection presented to Veterinary Clinical Complex at LUVAS, Hisar. A history of tick infestation and clinical indication of hemoparasitic disease, which include fever, anorexia, fatigue, corneal opacity, malena, cutaneous petechiae and ecchymoses, epistaxis, cachexia, lymph adenomegaly, pale mucous membranes, etc., were considered while choosing the dogs. Because clinical signs overlap among various hemoparasites, we examined dogs using individual PCR for four hemoparasites: B. vogeli, B. gibsoni H. canis and E. canis, PCR was conducted using Dream Taq Green PCR Master Mix (Thermo Scientific, USA) and the primers used were E. canis (F  ECAN 5 and R  HE3), H. canis (HEP-F and HEP-R), B. vogeli (F PIRO-A1 and R PIRO-B) and B. gibsoni (F Gib 599 and R Gib 1270) (Bhagwan et al., 2024).  The PCR conditions included an initial denaturation at 94 °C for 2 minutes, then 34 cycles of denaturation (94 °C for 1 minute), annealing (1 minute) and extension (72 °C for 1 kb/min) followed by 6-minute extension at 72 °C. Of these, 17 dogs were detected positive for E. canis alone (Bhagwan et al., 2024). The selected dogs were analyzed in the present study. During the clinical investigation, complete clinical history, including breed, age, sex and nature of illness, was collected for each dog. Thereafter, the vital parameters (rectal temperature (°F), pulse rate (per minute) and respiration rate (per minute) were measured and complete hematological and biochemical analyses were performed. The superficial lymph nodes were palpated. A total of approximate 6 ml blood was collected from each dog. One millilitre of blood was transferred into two EDTA tubes for haematological examination and DNA extraction. The remaining 4 mL of blood was poured into a plain tube containing a clot activator to facilitate serum separation.
       
Blood samples collected in K3EDTA-coated tubes were subjected to complete haematological analysis using an automated haematology cell counter (MS4s, Melet Schlosing Lab). Erythrocytic indices assessed included haemoglobin (Hb, g/dl), mean corpuscular haemoglobin concentration (MCHC, g/dl), hematocrit (Hct, %), mean corpuscular volume (MCV, fl), mean corpuscular haemoglobin (MCH, pg) and total erythrocyte count (TEC, M/mm3). Additionally, total leucocyte count (TLC), monocytes, lymphocytes, neutrophils, basophils and eosinophils, all expressed as percentages, as well as thrombocyte count (THR) were also analyzed.
       
The biochemical profiles were analyzed from serum samples using a automated random-access clinical chemistry analyzer (EM Destiny 180, Erba Diagnostics Mannheim GmbH). The liver function was measured by analysing serum total, direct and indirect bilirubin (mg/dl), alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), gamma-glutamyl transferase (GGT, U/L) and alkaline phosphatase (U/L). The protein profile consisted of total protein, albumin, globulin in g/dl and the albumin to globulin ratio (A/G). For kidney function, creatinine (mg/dl) and blood urea nitrogen (BUN, mg/dl) were measured.
       
Of the 17 E. canis-positive samples by PCR of 16s rRNA (Bhagwan et al., 2024), two PCR products were gel-purified and sequenced from both ends. The sequencing results were used to identify sequence similarity with the E. canis sequence retrieved from NCBI by phylogenetic analysis using MEGA X software (Kumar et al., 2018). The E. canis sequences from present study were aligned along with Ehrlichia spp. and Anaplasma platys sequences retrieved from NCBI using CLUSTALW program. The aligned sequences were used to generate a phylogenetic tree using the Maximum Likelihood method with the Kimura 2-parameter model, keeping bootstrap values to 1000 replicates, where Anaplasma platys (MG712286) was used as the outgroup. Itol software was used to visualize the phylogenetic tree (Letunic and Bork, 2024).
       
The data generated were analyzed using a statistical software package (SPSS 23.1). Various parameters reported in CME detected dogs were compared to those observed in healthy ones (Fielder, 2022: Kaneko, 2008). Results are reported as mean ± S.E. with a 0.05 level of significance.

RESULTS AND DISCUSSION

Of 120 suspected dogs, 17 (14.16%) were positive for E. canis by microscopic examination (Fig 1) and 16S rRNA PCR assay (Bhagwan et al., 2024). While analyzing breed-wise variation of CME in dogs, the majority of cases were reported in non-descript breeds, followed by Labrador retriever and German shepherd, Bully (Fig 2a). The sex-wise occurrence of CME revealed almost equal detection rates in male and female dogs (Fig 2b). The majority of CME cases were diagnosed in dogs over a year old. (Fig 2c).

Fig 1: Photomicrograph showing Ehrlichia canis morula inside monocyte (Giemsa stain, 1000x).



Fig 2: Distribution of E. canis in different breeds, age and sex.


       
The clinical profiles of dogs detected with E. canis are shown in Table 1 and Fig 3. Pyrexia (82.35%), anorexia (70.58%), congested mucous membrane (41.17%), pale mucous membrane (58.82%) and lethargy (41.17%) were the most prevalent clinical symptoms linked to CME. Ticks (Rhipicephalus sanguineus) were present on 76.47% of dogs that detected positive for E. canis.

Fig 3: Clinical finding of dogs detected with E. canis (a) Tick infestation; (b) Corneal opacity; c) Epistaxis; (d) Icterus.



Table 1: Clinical traits associated with CME (n = 17).


       
The hematological profiles of CME affected dogs (n=17) are provided in Table 2. The Hb levels in the most of dogs affected with CME were below the normal reference range. The TEC was within normal limits (n=9), followed by values below the lower limit (n=8). PCV was less than normal limit of the reference range in most cases (n=15). MCV (n=15), MCHC (n=9) and MCH (n=12) were below the lower limit of the normal reference range in most cases. The TLC was within normal limits in the most of the cases (n=10); however, the lymphocyte count was within normal limits in nine cases, followed by lymphocytosis. Neutrophils were within normal limits in the majority of CME affected dogs (n=15). Eosinophil count, basophil count and monocyte were detected within normal limits in most cases. Thrombocytopenia was reported in all CME positive cases. Overall blood profile found microcytic hypochromic to normochromic anemia, thrombocytopenia, normal to elevated TLC with absolute neutrophilia and normal to elevated lymphocyte count.

Table 2: Erythrocytic indices of dogs affected with CME (n=17).


       
Table 3 depicts the serum biochemical profiles of dogs with CME. Total protein levels in affected dogs were within normal limits in many cases (n=10). There was hypoalbuminemia in all cases and the A:G ratio was found below the lower limit in most cases (n=16). GGT activity was above the upper limit in all cases. Urea level in serum was within the normal range (n=15), whereas serum creatinine level was above the upper limit in all cases. The total bilirubin level in affected dogs was within normal limits in the majority of cases (n=15). Serum direct bilirubin was within normal limits (n=15), whereas indirect bilirubin was within normal limits in all cases.

Table 3: Serum biochemical profile of dogs affected with CME (n=17).


       
Of the 17 positive samples, two randomly selected PCR products were sequenced. The obtained nucleotide sequences were submitted to the NCBI and accession numbers PX736498 and PX736499 were obtained. After phylogenetic analysis, both sequences from the present study clustered with E. canis sequences reported from different parts of the world (Fig 4). This confirmed the molecular identification of our sequence as E. canis. The sequences from the present study (PX736498 and PX736499) formed a separate cluster that was closely related to PX591180 from South Africa, with sequence identities of 94.18% and 86.43%, respectively. PX736498 and PX736499 had a high nucleotide identity (84.80%) with previously published E. canis sequences from India, Taiwan, Iraq, Brazil, Turkey, Japan, China and the USA.

Fig 4: Phylogenetic tree of 16s rRNA sequence of Ehrlichiae species and Anaplasma platys as out group.


       
In the present study, the most common clinical findings with CME were fever, anorexia, pale to congested mucous membrane, lethargy, lymphadenopathy and epistaxis, etc. Diverse clinical signs recorded in dogs with CME have been reported in previous studies (Harrus and Waner, 2011; Mylonakis et al., 2019), which indicate multi-organ dysfunctions due to underlying pathological changes such as vasculitis (Mylonakis et al., 2019). Haemorrhages manifested as epistaxis, dermal petechiae and ecchymoses, haematuria and melena in cases of CME during the present study had also been observed earlier (Rungsipipat et al., 2009; Bai et al., 2017; Chakraborty et al., 2024). The increased bleeding tendencies could be attributed to thrombocytopenia and the deposition of immune complexes on the vascular wall (Rungsipipat et al., 2009).
       
The present study reported more predisposition of non-descript breed (20%), followed by German shepherd (10.34%) and Labrador retriever (9.67%) for CME. In a previous study from Hisar, Haryana, non-descript dogs were more susceptible to the haemoprotozoan infections like E. canis (Bhagwan et al., 2024). Out of the known breeds, German Shepherds were found to be most frequently reported with E. canis as reported from other parts of India (Bhardwaj, 2013; Bhadesiya and Raval, 2015). When comparing CME in different age groups, the maximum number of cases was reported in dogs above one year of age, as reported earlier (Kottadamane et al., 2017; Singh et al., 2021). However, in a few studies, younger dogs (below 1 year) were more susceptible to CME (Rungsipipat et al., 2009; Chandrasekar et al., 2002). The present study reported an almost equal detection rate of CME in male and female dogs as reported earlier (Revathi et al., 2025; Rungsipipat et al., 2009).
       
Haematological profile revealed a lower level of Hb and total erythrocyte count in CME-affected dogs which are in agreement with published reports (Srikala et al., 2012; Singh et al., 2021; Chakraborty et al., 2024). These alterations could be due to the higher affinity of E. canis towards the vascular endothelial cells, leading to vasculitis, which is manifested as multiple petechial or ecchymotic haemorrhages (Sainz et al., 2015; Mohanapriya et al., 2021). In the majority of CME-affected dogs, the levels of PCV (n=15), MCV (n=15) and MCH (n=12) were found below the lower limit. This suggests the microcytic hypochromic to normochromic anemia which is in contrast to the usual finding of normocytic normochromic anaemia (Nakaghi et al., 2008; Silva et al., 2012). The destruction of canine RBC may also be due to an immunological mechanism, resulting in haemoglobinuria and anaemia (Taylor et al., 2015).
       
Thrombocyte count was found to be drastically reduced, which is the characteristic feature of CME (Woody and Hoskins, 1991; Mylonakis et al., 2017). The reports of thrombocytopenia during ehrlichiosis had been consistently reported earlier (Waner et al., 2001; Nakaghi et al., 2008; Chakraborty et al., 2024). Thrombocytopenia, mainly due to large-scale destruction of the cells in the spleen that begins a few days after the infection (Smith et al., 1975), alongside bone marrow hypoplasia, are also amongst the primary causes of pancytopenia and thrombocytopenia (Waner, 2008). Increased splenic sequestration, platelet consumption and decreased platelet lifespan may be the possible factors for thrombocytopenia (Harrus et al., 1999). Reduced synthesis of coagulation proteins in the liver due to necrosis may also cause increased platelet aggregation, further aggravating the thrombocytopenia (Mylonakis et al., 2019).                
The TLC was found within normal limits in the majority of the cases (n=10) as reported earlier (Rungsipipat et al., 2009). Leukocytosis in the present study is in agreement with a study conducted from Maharastra, India (Parmar et al., 2013). However, in a study from Israel, significant leucopenia was documented in dogs affected with E. canis (Waner et al., 2008). Lymphocyte count was within normal limits in more than 50% of cases (n=9), followed by absolute lymphocytosis, which is in agreement with published literature (Parmar et al., 2013). The possible reasons for lymphocytosis may be due to chronic immune stimulation, lymphaenopathy and lymphoid hyperplasia (Tamai and Staji, 2025). Neutrophil count was found within normal limits in the majority of the cases (n=15); however, previous studies had reported neutrophilia in dogs affected with E. canis (Rungsipipat et al., 2009; Parmar et al., 2013).

Total protein level in serum of CME-affected dogs was within normal limits in the majority of the cases (n=10), followed by hypoprotenemia (n=7). CME affected dogs were reported with hypoalbuminemia in all the cases with A:G ratio below the lower limit in majority of dogs (n=16). Hypoproteinemia and hyperglobulinemia associated with ehrlichiosis in dogs were in agreement with previous study reports (Rungsipipat et al., 2009; Srikala et al., 2012; Bai et al., 2017), while a study had recorded a significant increase in total protein levels in dogs with ehrlichiosis (Bhardwaj, 2013). The probable causes for hypoproteinemia and hypoalbuminemia are loss of plasma proteins from the vascular endothelial pores due to vasculitis (Woody and Hoskins, 1991) or due to reduced protein synthesis as a result of concurrent liver diseases (Reardon and Pierce, 1981) or glomerulonephritis (Mylonakis et al., 2010). Liver function tests (ALT, AST and ALP) in affected dogs were within normal limits in the majority of the cases. However, studies from previous studies reported elevated levels of ALT, AST and ALP. However elevated level of GGT has been consistently reported in present and previous studies (Bhardwaj 2013; Srikala 2012). These findings of measured GGT could be attributed to E. canis-induced inflammation of the sinusoidal endothelium and infiltration of perivascular mononuclear cells in liver (Mylonakis et al., 2010; Nair et al., 2016).
       
Serum urea was found within normal limits (n=15) in the majority of CME affected dog, while serum creatinine level was found above the upper limit in all the cases, as reported earlier (Srivastava and Srivastava, 2011). The results were in agreement with studies reporting renal damage due to concurrent immune-mediated glomerulonephritis caused by E. canis antigen against the basement membrane of the glomerulus (Silva et al., 2016).
       
The phylogenetic analysis based on 16S rRNA sequence confirmed the genetic identity of the PCR product as E. canis. Similar methods for species identification have been used previously (Parthiban et al., 2024; Kopparthi et al., 2025). Phylogenetic analysis revealed that the E. canis from the present study were closely related and were phylogenetically similar to sequences from South Africa, India, Taiwan, Iraq, Brazil, Turkey, Japan, China and the USA, with high genetic identity (Supplementary Fig 1), implying a regionally conserved nature of the 16s rRNA sequence E. canis (Nguyen et al., 2025; Venugopal et al., 2026).

Supplementary Fig 1: Heat map depicting percent nucleotide identity between the analysed 16s rRNA sequence of Ehrlichiae species and Anaplasma platys.

CONCLUSION

The study concluded that these findings could facilitate the diagnosis of canine monocytic ehrlichiosis (CME). Phylogenetic analysis demonstrated a high sequence identity between isolates of E. canis from the present study and those from South Africa, India, Taiwan, Iraq, Brazil, Turkey, Japan, China and the USA, thereby confirming their identity. These findings demonstrate the importance of haematological and biochemical profiling for diagnosing and managing E. canis infections. The study also highlights the necessity for greater awareness and improved control measures for this neglected tick-borne disease in dogs.

ACKNOWLEDGEMENT

The authors are grateful to the Department of Veterinary Clinical Complex, LUVAS, Hisar, for extending supporting terms through the availability of all clinical and laboratory facilities for this research work.
 
Disclaimers
 
The views and conclusions expressed in this article are exclusively those of the authors and do not necessarily reflect the views of their institutions. The authors are responsible for the quality and completeness of the information that was provided, but they bear no liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
The Institutional Animal Ethics Committee of Lala Lajpat Rai University of Veterinary and Animal Sciences in Hisar, Haryana, India, granted approval for all animal procedures for studies and handling techniques. 

CONFLICT OF INTEREST

The authors state that there are no conflicts of interest related to the publication of this work. The study’s design, sample collection, analysis, publication decision and article preparation were all independent of funding or sponsorship.

REFERENCES


  1. Bai, L., Goel, P., Jhambh, R., Kumar, P. and Joshi, V.G. (2017). Molecular prevalence and haemato-biochemical profile of canine monocytic ehrlichiosis in dogs in and around Hisar, Haryana, India. Journal of Parasitic Diseases. 41(3): 647-654.

  2. Bhadesiya, C.M. and Raval, S.K. (2015). Hematobiochemical changes in ehrlichiosis in dogs of Anand region, Gujarat. Veterinary World. 8(6): 713-717.

  3. Bhagwan, J., Singh, Y., Jhambh, R., Chaudhari, M. and Kumar, P. (2024). Molecular and microscopic detection of haemoprotozoan diseases in dogs from Haryana, India. Parasitology Research. 123(10): 354.

  4. Bhardwaj, R.K. (2013). Therapeutic management of acute canine monocytic ehrlichiosis. Indian Veterinary Journal. 90(2): 138-139.

  5. Chakraborty, A., Rath, P.K., Panda, S.K. et al. (2024). Molecular confirmation, epidemiology and pathophysiology of Ehrlichia canis prevalence in eastern India. Pathogens. 13(9): 803.

  6. Chandrasekar, M., Nambi, A.P., Ramprabhu, R. and Dhanapalan, P. (2002). Epizootiological studies on canine ehrlichiosis. Indian Veterinary Journal. 79: 85-86.

  7. Fielder, S.E. (2022). Reference guides. In: Aiello, S.E. and Moses, M.A. (Eds.), The Merck Veterinary Manual. Merck and Co. Inc., Rahway.

  8. Harrus, S. and Waner, T. (2011). Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): An overview. The Veterinary Journal. 187(3): 292-296.

  9. Harrus, S., Waner, T., Bark, H., Jongejan, F. and Cornelissen, A.W. (1999). Recent advances in pathogenesis of canine monocytic ehrlichiosis. Journal of Clinical Microbiology. 37(9): 2745-2749.

  10. Kaneko, J.J., Harvey, J.W. and Bruss, M.L. (2008). Clinical Biochemistry of Domestic Animals. Academic Press, San Diego.

  11. Kopparthi, J., Chennuru, C.S., Vukka, C.R. and Devalum, R.P. (2025). Prevalence, molecular identification and phylogenic analysis of Hepatozoon canis from naturally infected dogs in Andhra Pradesh, South India. Indian Journal of Animal Research. 1-5. doi: 10.18805/IJAR.B-5420.

  12. Kottadamane, M.R., Dhaliwal, P.S., Singla, L.D., Bansal, B.K. and Uppal, S.K. (2017). Clinical and hematobiochemical response in canine monocytic ehrlichiosis seropositive dogs of Punjab. Veterinary World. 10(2): 255-261.

  13. Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 35(6): 1547- 1549.

  14. Letunic, I. and Bork, P. (2024). Interactive Tree of Life (iTOL) v6: Recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research. 52(W1): W78-W82.

  15. Megat Abd Rani, P.A., Irwin, P.J., Gatne, M., Coleman, G.T. and Traub, R.J. (2010). Canine vector-borne diseases in India: A review of the literature and identification of existing knowledge gaps. Parasites and Vectors. 3(1): 28.

  16. Mohanapriya, T., Ramprabhu, R., Kumar, V., Enbavelan, P.A. and Ganesan, A. (2021). Clinico-pathological diagnosis of transmissible venereal tumour with Hepatozoon canis and Babesia canis Infection in a Chippiparai Dog. Indian Journal of Animal Research. 55(9): 1121-1124. doi: 10.18805/IJAR.B-4168.

  17. Mylonakis, M.E. and Theodorou, K.N. (2017). Canine monocytic ehrlichiosis: An update on diagnosis and treatment. Acta Veterinaria Beograd. 67(3): 299-317.

  18. Mylonakis, M.E., Harrus, S. and Breitschwerdt, E.B. (2019). An update on the treatment of canine monocytic ehrlichiosis (Ehrlichia canis). The Veterinary Journal. 246: 45-53.

  19. Mylonakis, M.E., Koutinas, A.F., Billinis, C., et al. (2003). Evaluation of cytology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): A comparison between five methods. Veterinary Microbiology. 91(2-3): 197-204.

  20. Mylonakis, M.E., Kritsepi-Konstantinou, M., Dumler, J.S. et al. (2010). Severe hepatitis associated with acute Ehrlichia canis infection in a dog. Journal of Veterinary Internal Medicine. 24(3): 633-638.

  21. Nair, A.D., Cheng, C., Ganta, C.K. et al. (2016). Comparative experimental infection study in dogs with Ehrlichia canis and related pathogens. PLoS ONE. 11(2): e0148239.

  22. Nakaghi, A.C., Machado, R.Z., Costa, M.T. andré, M.R. and Baldani, C.D. (2008). Canine ehrlichiosis: Clinical, hematological, serological and molecular aspects. Ciência Rural. 38: 766-770.

  23. Nambooppha, B., Rittipornlertrak, A., Tattiyapong, M., Tangtrongsup, S., Tiwananthagorn, S., Chung, Y.T. and Sthitmatee, N. (2018). Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant protein genes. Infection Genetics and Evolution. 63: 116-125.

  24. Nguyen, L.V., Van Nguyen, D. and Du, V.T. (2025). Molecular prevalence and 16S rRNA gene analysis of Ehrlichia canis in dogs in Ho Chi Minh City, Vietnam. World Veterinary Journal. 15(2): 264-273.

  25. Parmar, C., Pednekar, R., Jayraw, A. and Gatne, M. (2013). Comparative diagnostic methods for canine ehrlichiosis. Turkish Journal of Veterinary and Animal Sciences. 37(3): 282-290.

  26. Parthiban, R., Palavesam, A.B., Srinivasan, S. et al. (2024). Molecular characterization of Ehrlichia canis from naturally infected dogs in India. Research in Veterinary Science. 175: 105311.

  27. Reardon, M.J. and Pierce, K.R. (1981). Acute experimental canine ehrlichiosis I. Sequential reaction of hemic and lymphoreticular systems. Veterinary Pathology. 18(1): 48-61.

  28. Revathi, P., Bharathi, M.V., Madhanmohan, M., Latha, B.R. and Rani, K.V. (2025). Molecular epidemiology, characterisation of Hepatozoon canis in dogs as well as in ticks and haemato-biochemical profile of the infected dogs in Chennai. Indian Journal of Animal Research. 59(5): 852-858. doi: 10.18805/IJAR.B-4801.

  29. Rungsipipat, A., Oda, M., Kumpoosiri, N. et al. (2009). Clinicopathological study of experimentally induced canine monocytic ehrlichiosis. Comparative Clinical Pathology. 18(1): 13-22.

  30. Sainz, Á., Roura, X., Miró, G. et al. (2015). Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasites and Vectors. 8: 75.

  31. Sarma, K., Mondal, D., Saravanan, M. and Mahendran, K. (2015). Evaluation of haemato-biochemical and oxidative indices in naturally infected concomitant tick borne intracellular diseases in dogs. Asian Pacific Journal of Tropical Disease. 5(1): 60-66.

  32. Silva, G.C.F., Benitez, A.N., Girotto, A. et al. (2012). Occurrence of Ehrlichia canis and Anaplasma platys in household dogs from northern Paraná. Revista Brasileira de Parasitologia Veterinária. 21: 379-385.

  33. Silva, L.S., Pinho, F.A., Prianti, M.G. et al. (2016). Renal histopathological changes in dogs naturally infected with Ehrlichia canis. Brazilian Journal of Veterinary Pathology. 9(1): 2-15.

  34. Singh, J., Singh, R.S., Singh, H., Gupta, D.K. and Randhawa, S.S. (2021). Clinical and haemato-biochemical observations in dogs naturally infected with canine monocytic ehrlichiosis. Exploratory Animal and Medical Research. 11(2): 214- 219.

  35. Smith, R.D., Ristic, M., Huxsoll, D.L. and Baylor, R.A. (1975). Platelet kinetics in canine ehrlichiosis. Infection and Immunity. 11(6): 1216-1221.

  36. Srikala, D., Satish Kumar, K., Amruth Kumar, V.V. and Tirumala Rao, D.S. (2012). Clinical and therapeutic aspects of canine monocytic ehrlichiosis. Indian Journal of Veterinary Medicine. 32(2): 109-110.

  37. Srivastava, M.K. and Srivastava, A. (2011). Canine ehrlichiosis in dog. Indian Journal of Veterinary Medicine. 31(2): 128- 129.

  38. Stich, R.W., Rikihisa, Y., Ewing, S.A., Needham, G.R., Grover, D.L. and Jittapalapong, S. (2002). Detection of Ehrlichia canis in canine carrier blood and in individual experimentally infected ticks with a p30-based PCR assay. Journal of Clinical Microbiology. 40(2): 540-546.

  39. Tamai, I.A. and Staji, H. (2025). Molecular identification and associated risk factors of Hepatozoon canis among dogs visiting veterinary clinics in Tehran, Iran. BMC Veterinary Research. 21(1): 660.

  40. Taylor, M.A., Coop, R.L. and Wall, R. (2015). Veterinary Parasitology. John Wiley and Sons, Chichester.

  41. Venugopal, V., Deepa, P.M., Muhasin Asaf, V.N. et al. (2026). Molecular epidemiology and phylogenetics of canine haemoprotozoan and rickettsial infections in southern India. Veterinary Parasitology: Regional Studies and Reports. 67: 1013 99.

  42. Waner, T. (2008). Hematopathological changes in dogs infected with Ehrlichia canis. Israel Journal of Veterinary Medicine. 63(1): 19.

  43. Waner, T., Harrus, S., Jongejan, F., Bark, H., Keysary, A. and Cornelissen, A.W. (2001). Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis. Veterinary Parasitology. 95(1): 1-15.

  44. Woody, B.J. and Hoskins, J.D. (1991). Ehrlichial diseases of dogs. Veterinary Clinics of North America: Small Animal Practice. 21: 75-98.
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
41
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Molecular and Epidemiological Investigation with Haematobiochemical Profiling of Ehrlichia canis Infection in Dogs from Haryana, India

    J
    Jai Bhagwan1
    0000-0002-9509-4067
    Y
    Yudhbir Singh1
    0000-0001-5245-4731
    R
    Ricky Jhambh1
    0000-0001-8039-8140
    P
    Parveen Kumar2,*
    0000-0001-9627-3748
    R
    Ritik Kaul1
    M
    Mahavir Chaudhary3
    D
    Divya Agnihotri1
    0000-0002-0719-9281
    1Department of Veterinary Medicine, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.
    2Department of Veterinary Microbiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.
    3Department of Animal Genetics and Breeding, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.

    ABSTRACT

    Background: The study aimed to perform the molecular investigation, clinical observations and haemato-biochemical abnormalities associated with dogs naturally affected with canine monocytic ehrlichiosis (CME) from Haryana, India.

    Methods: The study included 120 dogs suspected of haemoparasitic infection. History and clinical profiles were noted for each dog. PCR tested for four haemoparasites: H. canis, E. canis, B. vogeli and B. gibsoni. Seventeen dogs positive for E. canis underwent clinical investigation, haematological and biochemical analyses. Two 16s rRNA PCR products were sequenced and phylogenetic trees were prepared using MEGA X software. Data were analysed using SPSS 23.1. Parameters in CME-detected animals were compared with those in healthy animals.

    Result: Most common clinical manifestations associated with CME were pyrexia, anorexia, a congested to pale mucous membrane and lethargy. Epistaxis, lymphadenomegaly, hindlimb weakness, melena and ocular discharge were among the less frequent clinical signs. Haematological findings revealed microcytic hypochromic to normochromic anaemia, thrombocytopenia and normal to raised leucocyte count with absolute neutrophilia and normal to raised lymphocyte count as the main observation. In affected dogs, serum biochemical profiles revealed normal to low protein levels with hypoalbuminemia, reduced A:G ratio, higher GGT activity and an elevated creatinine level. Phylogenetic analysis revealed that the partial 16s rRNA sequences of E. canis in the current study were similar to those of E. canis isolates from various regions of India and the world. The current study emphassis the significance of clinical, haematological and biochemical characteristics in the detection and treatment of dogs with CME.

    INTRODUCTION

    Ehrlichia belongs to the family Ehrlichiaceae, within the order Rickettsiales and the class Alphaproteobacteria. E. canis is commonly reported in India and causes classical canine monocytic ehrlichiosis (CME) (Megat Abd Rani et al., 2010). CME is a multisystemic disease, where acute form is characterised by lethargy, high fever, anorexia, lymphadenopathy, splenomegaly, epistaxis, dermal petechiae, ecchymoses and occasional tick infestation (Harrus and Waner, 2011). The chronic form presents with ulcerative stomatitis, necrotic glossitis, hind limb, scrotal oedema, pyoderma, icterus and central nervous system signs (Mylonakis and Theodorou, 2017). Microscopic detection of E. canis morula is a traditional and commonly used method (Mylonakis et al., 2003), but it lacks sensitivity during chronic and subclinical infections (Woody and Hoskins, 1991). Other common methods include serological assays like ELISA and IFA; however, these have low specificity and cannot differentiate between ongoing and past infections (Waner at al., 2001). Previous studies found that haemato-biochemical and clinical findings could aid early detection of E. canis in dogs (Bai et al., 2017; Sarma et al., 2015). Therefore, the current study aimed to investigate clinical and haemato-biochemical indicators for E. canis diagnosis in dogs. Previous studies have used PCR methods focusing on the 16s rRNA, p30 outer membrane protein, dsb and gp 19 gene regions (Stich et al., 2002; Nambooppha et al., 2018). In this study, we used the 16s rRNA region for PCR detection and genetic characterization of the organism.

    MATERIALS AND METHODS

    The Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS) Institutional Animal Ethics Committee approved the study’s sample collection and animal handling protocols on 25/03/2021 (VCC/IAEC/630-651). The present study was conducted on 120 dogs suspected to have haemoparasitic infection presented to Veterinary Clinical Complex at LUVAS, Hisar. A history of tick infestation and clinical indication of hemoparasitic disease, which include fever, anorexia, fatigue, corneal opacity, malena, cutaneous petechiae and ecchymoses, epistaxis, cachexia, lymph adenomegaly, pale mucous membranes, etc., were considered while choosing the dogs. Because clinical signs overlap among various hemoparasites, we examined dogs using individual PCR for four hemoparasites: B. vogeli, B. gibsoni H. canis and E. canis, PCR was conducted using Dream Taq Green PCR Master Mix (Thermo Scientific, USA) and the primers used were E. canis (F  ECAN 5 and R  HE3), H. canis (HEP-F and HEP-R), B. vogeli (F PIRO-A1 and R PIRO-B) and B. gibsoni (F Gib 599 and R Gib 1270) (Bhagwan et al., 2024).  The PCR conditions included an initial denaturation at 94 °C for 2 minutes, then 34 cycles of denaturation (94 °C for 1 minute), annealing (1 minute) and extension (72 °C for 1 kb/min) followed by 6-minute extension at 72 °C. Of these, 17 dogs were detected positive for E. canis alone (Bhagwan et al., 2024). The selected dogs were analyzed in the present study. During the clinical investigation, complete clinical history, including breed, age, sex and nature of illness, was collected for each dog. Thereafter, the vital parameters (rectal temperature (°F), pulse rate (per minute) and respiration rate (per minute) were measured and complete hematological and biochemical analyses were performed. The superficial lymph nodes were palpated. A total of approximate 6 ml blood was collected from each dog. One millilitre of blood was transferred into two EDTA tubes for haematological examination and DNA extraction. The remaining 4 mL of blood was poured into a plain tube containing a clot activator to facilitate serum separation.
           
    Blood samples collected in K3EDTA-coated tubes were subjected to complete haematological analysis using an automated haematology cell counter (MS4s, Melet Schlosing Lab). Erythrocytic indices assessed included haemoglobin (Hb, g/dl), mean corpuscular haemoglobin concentration (MCHC, g/dl), hematocrit (Hct, %), mean corpuscular volume (MCV, fl), mean corpuscular haemoglobin (MCH, pg) and total erythrocyte count (TEC, M/mm3). Additionally, total leucocyte count (TLC), monocytes, lymphocytes, neutrophils, basophils and eosinophils, all expressed as percentages, as well as thrombocyte count (THR) were also analyzed.
           
    The biochemical profiles were analyzed from serum samples using a automated random-access clinical chemistry analyzer (EM Destiny 180, Erba Diagnostics Mannheim GmbH). The liver function was measured by analysing serum total, direct and indirect bilirubin (mg/dl), alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), gamma-glutamyl transferase (GGT, U/L) and alkaline phosphatase (U/L). The protein profile consisted of total protein, albumin, globulin in g/dl and the albumin to globulin ratio (A/G). For kidney function, creatinine (mg/dl) and blood urea nitrogen (BUN, mg/dl) were measured.
           
    Of the 17 E. canis-positive samples by PCR of 16s rRNA (Bhagwan et al., 2024), two PCR products were gel-purified and sequenced from both ends. The sequencing results were used to identify sequence similarity with the E. canis sequence retrieved from NCBI by phylogenetic analysis using MEGA X software (Kumar et al., 2018). The E. canis sequences from present study were aligned along with Ehrlichia spp. and Anaplasma platys sequences retrieved from NCBI using CLUSTALW program. The aligned sequences were used to generate a phylogenetic tree using the Maximum Likelihood method with the Kimura 2-parameter model, keeping bootstrap values to 1000 replicates, where Anaplasma platys (MG712286) was used as the outgroup. Itol software was used to visualize the phylogenetic tree (Letunic and Bork, 2024).
           
    The data generated were analyzed using a statistical software package (SPSS 23.1). Various parameters reported in CME detected dogs were compared to those observed in healthy ones (Fielder, 2022: Kaneko, 2008). Results are reported as mean ± S.E. with a 0.05 level of significance.

    RESULTS AND DISCUSSION

    Of 120 suspected dogs, 17 (14.16%) were positive for E. canis by microscopic examination (Fig 1) and 16S rRNA PCR assay (Bhagwan et al., 2024). While analyzing breed-wise variation of CME in dogs, the majority of cases were reported in non-descript breeds, followed by Labrador retriever and German shepherd, Bully (Fig 2a). The sex-wise occurrence of CME revealed almost equal detection rates in male and female dogs (Fig 2b). The majority of CME cases were diagnosed in dogs over a year old. (Fig 2c).

    Fig 1: Photomicrograph showing Ehrlichia canis morula inside monocyte (Giemsa stain, 1000x).



    Fig 2: Distribution of E. canis in different breeds, age and sex.


           
    The clinical profiles of dogs detected with E. canis are shown in Table 1 and Fig 3. Pyrexia (82.35%), anorexia (70.58%), congested mucous membrane (41.17%), pale mucous membrane (58.82%) and lethargy (41.17%) were the most prevalent clinical symptoms linked to CME. Ticks (Rhipicephalus sanguineus) were present on 76.47% of dogs that detected positive for E. canis.

    Fig 3: Clinical finding of dogs detected with E. canis (a) Tick infestation; (b) Corneal opacity; c) Epistaxis; (d) Icterus.



    Table 1: Clinical traits associated with CME (n = 17).


           
    The hematological profiles of CME affected dogs (n=17) are provided in Table 2. The Hb levels in the most of dogs affected with CME were below the normal reference range. The TEC was within normal limits (n=9), followed by values below the lower limit (n=8). PCV was less than normal limit of the reference range in most cases (n=15). MCV (n=15), MCHC (n=9) and MCH (n=12) were below the lower limit of the normal reference range in most cases. The TLC was within normal limits in the most of the cases (n=10); however, the lymphocyte count was within normal limits in nine cases, followed by lymphocytosis. Neutrophils were within normal limits in the majority of CME affected dogs (n=15). Eosinophil count, basophil count and monocyte were detected within normal limits in most cases. Thrombocytopenia was reported in all CME positive cases. Overall blood profile found microcytic hypochromic to normochromic anemia, thrombocytopenia, normal to elevated TLC with absolute neutrophilia and normal to elevated lymphocyte count.

    Table 2: Erythrocytic indices of dogs affected with CME (n=17).


           
    Table 3 depicts the serum biochemical profiles of dogs with CME. Total protein levels in affected dogs were within normal limits in many cases (n=10). There was hypoalbuminemia in all cases and the A:G ratio was found below the lower limit in most cases (n=16). GGT activity was above the upper limit in all cases. Urea level in serum was within the normal range (n=15), whereas serum creatinine level was above the upper limit in all cases. The total bilirubin level in affected dogs was within normal limits in the majority of cases (n=15). Serum direct bilirubin was within normal limits (n=15), whereas indirect bilirubin was within normal limits in all cases.

    Table 3: Serum biochemical profile of dogs affected with CME (n=17).


           
    Of the 17 positive samples, two randomly selected PCR products were sequenced. The obtained nucleotide sequences were submitted to the NCBI and accession numbers PX736498 and PX736499 were obtained. After phylogenetic analysis, both sequences from the present study clustered with E. canis sequences reported from different parts of the world (Fig 4). This confirmed the molecular identification of our sequence as E. canis. The sequences from the present study (PX736498 and PX736499) formed a separate cluster that was closely related to PX591180 from South Africa, with sequence identities of 94.18% and 86.43%, respectively. PX736498 and PX736499 had a high nucleotide identity (84.80%) with previously published E. canis sequences from India, Taiwan, Iraq, Brazil, Turkey, Japan, China and the USA.

    Fig 4: Phylogenetic tree of 16s rRNA sequence of Ehrlichiae species and Anaplasma platys as out group.


           
    In the present study, the most common clinical findings with CME were fever, anorexia, pale to congested mucous membrane, lethargy, lymphadenopathy and epistaxis, etc. Diverse clinical signs recorded in dogs with CME have been reported in previous studies (Harrus and Waner, 2011; Mylonakis et al., 2019), which indicate multi-organ dysfunctions due to underlying pathological changes such as vasculitis (Mylonakis et al., 2019). Haemorrhages manifested as epistaxis, dermal petechiae and ecchymoses, haematuria and melena in cases of CME during the present study had also been observed earlier (Rungsipipat et al., 2009; Bai et al., 2017; Chakraborty et al., 2024). The increased bleeding tendencies could be attributed to thrombocytopenia and the deposition of immune complexes on the vascular wall (Rungsipipat et al., 2009).
           
    The present study reported more predisposition of non-descript breed (20%), followed by German shepherd (10.34%) and Labrador retriever (9.67%) for CME. In a previous study from Hisar, Haryana, non-descript dogs were more susceptible to the haemoprotozoan infections like E. canis (Bhagwan et al., 2024). Out of the known breeds, German Shepherds were found to be most frequently reported with E. canis as reported from other parts of India (Bhardwaj, 2013; Bhadesiya and Raval, 2015). When comparing CME in different age groups, the maximum number of cases was reported in dogs above one year of age, as reported earlier (Kottadamane et al., 2017; Singh et al., 2021). However, in a few studies, younger dogs (below 1 year) were more susceptible to CME (Rungsipipat et al., 2009; Chandrasekar et al., 2002). The present study reported an almost equal detection rate of CME in male and female dogs as reported earlier (Revathi et al., 2025; Rungsipipat et al., 2009).
           
    Haematological profile revealed a lower level of Hb and total erythrocyte count in CME-affected dogs which are in agreement with published reports (Srikala et al., 2012; Singh et al., 2021; Chakraborty et al., 2024). These alterations could be due to the higher affinity of E. canis towards the vascular endothelial cells, leading to vasculitis, which is manifested as multiple petechial or ecchymotic haemorrhages (Sainz et al., 2015; Mohanapriya et al., 2021). In the majority of CME-affected dogs, the levels of PCV (n=15), MCV (n=15) and MCH (n=12) were found below the lower limit. This suggests the microcytic hypochromic to normochromic anemia which is in contrast to the usual finding of normocytic normochromic anaemia (Nakaghi et al., 2008; Silva et al., 2012). The destruction of canine RBC may also be due to an immunological mechanism, resulting in haemoglobinuria and anaemia (Taylor et al., 2015).
           
    Thrombocyte count was found to be drastically reduced, which is the characteristic feature of CME (Woody and Hoskins, 1991; Mylonakis et al., 2017). The reports of thrombocytopenia during ehrlichiosis had been consistently reported earlier (Waner et al., 2001; Nakaghi et al., 2008; Chakraborty et al., 2024). Thrombocytopenia, mainly due to large-scale destruction of the cells in the spleen that begins a few days after the infection (Smith et al., 1975), alongside bone marrow hypoplasia, are also amongst the primary causes of pancytopenia and thrombocytopenia (Waner, 2008). Increased splenic sequestration, platelet consumption and decreased platelet lifespan may be the possible factors for thrombocytopenia (Harrus et al., 1999). Reduced synthesis of coagulation proteins in the liver due to necrosis may also cause increased platelet aggregation, further aggravating the thrombocytopenia (Mylonakis et al., 2019).                
    The TLC was found within normal limits in the majority of the cases (n=10) as reported earlier (Rungsipipat et al., 2009). Leukocytosis in the present study is in agreement with a study conducted from Maharastra, India (Parmar et al., 2013). However, in a study from Israel, significant leucopenia was documented in dogs affected with E. canis (Waner et al., 2008). Lymphocyte count was within normal limits in more than 50% of cases (n=9), followed by absolute lymphocytosis, which is in agreement with published literature (Parmar et al., 2013). The possible reasons for lymphocytosis may be due to chronic immune stimulation, lymphaenopathy and lymphoid hyperplasia (Tamai and Staji, 2025). Neutrophil count was found within normal limits in the majority of the cases (n=15); however, previous studies had reported neutrophilia in dogs affected with E. canis (Rungsipipat et al., 2009; Parmar et al., 2013).

    Total protein level in serum of CME-affected dogs was within normal limits in the majority of the cases (n=10), followed by hypoprotenemia (n=7). CME affected dogs were reported with hypoalbuminemia in all the cases with A:G ratio below the lower limit in majority of dogs (n=16). Hypoproteinemia and hyperglobulinemia associated with ehrlichiosis in dogs were in agreement with previous study reports (Rungsipipat et al., 2009; Srikala et al., 2012; Bai et al., 2017), while a study had recorded a significant increase in total protein levels in dogs with ehrlichiosis (Bhardwaj, 2013). The probable causes for hypoproteinemia and hypoalbuminemia are loss of plasma proteins from the vascular endothelial pores due to vasculitis (Woody and Hoskins, 1991) or due to reduced protein synthesis as a result of concurrent liver diseases (Reardon and Pierce, 1981) or glomerulonephritis (Mylonakis et al., 2010). Liver function tests (ALT, AST and ALP) in affected dogs were within normal limits in the majority of the cases. However, studies from previous studies reported elevated levels of ALT, AST and ALP. However elevated level of GGT has been consistently reported in present and previous studies (Bhardwaj 2013; Srikala 2012). These findings of measured GGT could be attributed to E. canis-induced inflammation of the sinusoidal endothelium and infiltration of perivascular mononuclear cells in liver (Mylonakis et al., 2010; Nair et al., 2016).
           
    Serum urea was found within normal limits (n=15) in the majority of CME affected dog, while serum creatinine level was found above the upper limit in all the cases, as reported earlier (Srivastava and Srivastava, 2011). The results were in agreement with studies reporting renal damage due to concurrent immune-mediated glomerulonephritis caused by E. canis antigen against the basement membrane of the glomerulus (Silva et al., 2016).
           
    The phylogenetic analysis based on 16S rRNA sequence confirmed the genetic identity of the PCR product as E. canis. Similar methods for species identification have been used previously (Parthiban et al., 2024; Kopparthi et al., 2025). Phylogenetic analysis revealed that the E. canis from the present study were closely related and were phylogenetically similar to sequences from South Africa, India, Taiwan, Iraq, Brazil, Turkey, Japan, China and the USA, with high genetic identity (Supplementary Fig 1), implying a regionally conserved nature of the 16s rRNA sequence E. canis (Nguyen et al., 2025; Venugopal et al., 2026).

    Supplementary Fig 1: Heat map depicting percent nucleotide identity between the analysed 16s rRNA sequence of Ehrlichiae species and Anaplasma platys.

    CONCLUSION

    The study concluded that these findings could facilitate the diagnosis of canine monocytic ehrlichiosis (CME). Phylogenetic analysis demonstrated a high sequence identity between isolates of E. canis from the present study and those from South Africa, India, Taiwan, Iraq, Brazil, Turkey, Japan, China and the USA, thereby confirming their identity. These findings demonstrate the importance of haematological and biochemical profiling for diagnosing and managing E. canis infections. The study also highlights the necessity for greater awareness and improved control measures for this neglected tick-borne disease in dogs.

    ACKNOWLEDGEMENT

    The authors are grateful to the Department of Veterinary Clinical Complex, LUVAS, Hisar, for extending supporting terms through the availability of all clinical and laboratory facilities for this research work.
     
    Disclaimers
     
    The views and conclusions expressed in this article are exclusively those of the authors and do not necessarily reflect the views of their institutions. The authors are responsible for the quality and completeness of the information that was provided, but they bear no liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    The Institutional Animal Ethics Committee of Lala Lajpat Rai University of Veterinary and Animal Sciences in Hisar, Haryana, India, granted approval for all animal procedures for studies and handling techniques. 

    CONFLICT OF INTEREST

    The authors state that there are no conflicts of interest related to the publication of this work. The study’s design, sample collection, analysis, publication decision and article preparation were all independent of funding or sponsorship.

    REFERENCES


    1. Bai, L., Goel, P., Jhambh, R., Kumar, P. and Joshi, V.G. (2017). Molecular prevalence and haemato-biochemical profile of canine monocytic ehrlichiosis in dogs in and around Hisar, Haryana, India. Journal of Parasitic Diseases. 41(3): 647-654.

    2. Bhadesiya, C.M. and Raval, S.K. (2015). Hematobiochemical changes in ehrlichiosis in dogs of Anand region, Gujarat. Veterinary World. 8(6): 713-717.

    3. Bhagwan, J., Singh, Y., Jhambh, R., Chaudhari, M. and Kumar, P. (2024). Molecular and microscopic detection of haemoprotozoan diseases in dogs from Haryana, India. Parasitology Research. 123(10): 354.

    4. Bhardwaj, R.K. (2013). Therapeutic management of acute canine monocytic ehrlichiosis. Indian Veterinary Journal. 90(2): 138-139.

    5. Chakraborty, A., Rath, P.K., Panda, S.K. et al. (2024). Molecular confirmation, epidemiology and pathophysiology of Ehrlichia canis prevalence in eastern India. Pathogens. 13(9): 803.

    6. Chandrasekar, M., Nambi, A.P., Ramprabhu, R. and Dhanapalan, P. (2002). Epizootiological studies on canine ehrlichiosis. Indian Veterinary Journal. 79: 85-86.

    7. Fielder, S.E. (2022). Reference guides. In: Aiello, S.E. and Moses, M.A. (Eds.), The Merck Veterinary Manual. Merck and Co. Inc., Rahway.

    8. Harrus, S. and Waner, T. (2011). Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): An overview. The Veterinary Journal. 187(3): 292-296.

    9. Harrus, S., Waner, T., Bark, H., Jongejan, F. and Cornelissen, A.W. (1999). Recent advances in pathogenesis of canine monocytic ehrlichiosis. Journal of Clinical Microbiology. 37(9): 2745-2749.

    10. Kaneko, J.J., Harvey, J.W. and Bruss, M.L. (2008). Clinical Biochemistry of Domestic Animals. Academic Press, San Diego.

    11. Kopparthi, J., Chennuru, C.S., Vukka, C.R. and Devalum, R.P. (2025). Prevalence, molecular identification and phylogenic analysis of Hepatozoon canis from naturally infected dogs in Andhra Pradesh, South India. Indian Journal of Animal Research. 1-5. doi: 10.18805/IJAR.B-5420.

    12. Kottadamane, M.R., Dhaliwal, P.S., Singla, L.D., Bansal, B.K. and Uppal, S.K. (2017). Clinical and hematobiochemical response in canine monocytic ehrlichiosis seropositive dogs of Punjab. Veterinary World. 10(2): 255-261.

    13. Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 35(6): 1547- 1549.

    14. Letunic, I. and Bork, P. (2024). Interactive Tree of Life (iTOL) v6: Recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research. 52(W1): W78-W82.

    15. Megat Abd Rani, P.A., Irwin, P.J., Gatne, M., Coleman, G.T. and Traub, R.J. (2010). Canine vector-borne diseases in India: A review of the literature and identification of existing knowledge gaps. Parasites and Vectors. 3(1): 28.

    16. Mohanapriya, T., Ramprabhu, R., Kumar, V., Enbavelan, P.A. and Ganesan, A. (2021). Clinico-pathological diagnosis of transmissible venereal tumour with Hepatozoon canis and Babesia canis Infection in a Chippiparai Dog. Indian Journal of Animal Research. 55(9): 1121-1124. doi: 10.18805/IJAR.B-4168.

    17. Mylonakis, M.E. and Theodorou, K.N. (2017). Canine monocytic ehrlichiosis: An update on diagnosis and treatment. Acta Veterinaria Beograd. 67(3): 299-317.

    18. Mylonakis, M.E., Harrus, S. and Breitschwerdt, E.B. (2019). An update on the treatment of canine monocytic ehrlichiosis (Ehrlichia canis). The Veterinary Journal. 246: 45-53.

    19. Mylonakis, M.E., Koutinas, A.F., Billinis, C., et al. (2003). Evaluation of cytology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): A comparison between five methods. Veterinary Microbiology. 91(2-3): 197-204.

    20. Mylonakis, M.E., Kritsepi-Konstantinou, M., Dumler, J.S. et al. (2010). Severe hepatitis associated with acute Ehrlichia canis infection in a dog. Journal of Veterinary Internal Medicine. 24(3): 633-638.

    21. Nair, A.D., Cheng, C., Ganta, C.K. et al. (2016). Comparative experimental infection study in dogs with Ehrlichia canis and related pathogens. PLoS ONE. 11(2): e0148239.

    22. Nakaghi, A.C., Machado, R.Z., Costa, M.T. andré, M.R. and Baldani, C.D. (2008). Canine ehrlichiosis: Clinical, hematological, serological and molecular aspects. Ciência Rural. 38: 766-770.

    23. Nambooppha, B., Rittipornlertrak, A., Tattiyapong, M., Tangtrongsup, S., Tiwananthagorn, S., Chung, Y.T. and Sthitmatee, N. (2018). Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant protein genes. Infection Genetics and Evolution. 63: 116-125.

    24. Nguyen, L.V., Van Nguyen, D. and Du, V.T. (2025). Molecular prevalence and 16S rRNA gene analysis of Ehrlichia canis in dogs in Ho Chi Minh City, Vietnam. World Veterinary Journal. 15(2): 264-273.

    25. Parmar, C., Pednekar, R., Jayraw, A. and Gatne, M. (2013). Comparative diagnostic methods for canine ehrlichiosis. Turkish Journal of Veterinary and Animal Sciences. 37(3): 282-290.

    26. Parthiban, R., Palavesam, A.B., Srinivasan, S. et al. (2024). Molecular characterization of Ehrlichia canis from naturally infected dogs in India. Research in Veterinary Science. 175: 105311.

    27. Reardon, M.J. and Pierce, K.R. (1981). Acute experimental canine ehrlichiosis I. Sequential reaction of hemic and lymphoreticular systems. Veterinary Pathology. 18(1): 48-61.

    28. Revathi, P., Bharathi, M.V., Madhanmohan, M., Latha, B.R. and Rani, K.V. (2025). Molecular epidemiology, characterisation of Hepatozoon canis in dogs as well as in ticks and haemato-biochemical profile of the infected dogs in Chennai. Indian Journal of Animal Research. 59(5): 852-858. doi: 10.18805/IJAR.B-4801.

    29. Rungsipipat, A., Oda, M., Kumpoosiri, N. et al. (2009). Clinicopathological study of experimentally induced canine monocytic ehrlichiosis. Comparative Clinical Pathology. 18(1): 13-22.

    30. Sainz, Á., Roura, X., Miró, G. et al. (2015). Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasites and Vectors. 8: 75.

    31. Sarma, K., Mondal, D., Saravanan, M. and Mahendran, K. (2015). Evaluation of haemato-biochemical and oxidative indices in naturally infected concomitant tick borne intracellular diseases in dogs. Asian Pacific Journal of Tropical Disease. 5(1): 60-66.

    32. Silva, G.C.F., Benitez, A.N., Girotto, A. et al. (2012). Occurrence of Ehrlichia canis and Anaplasma platys in household dogs from northern Paraná. Revista Brasileira de Parasitologia Veterinária. 21: 379-385.

    33. Silva, L.S., Pinho, F.A., Prianti, M.G. et al. (2016). Renal histopathological changes in dogs naturally infected with Ehrlichia canis. Brazilian Journal of Veterinary Pathology. 9(1): 2-15.

    34. Singh, J., Singh, R.S., Singh, H., Gupta, D.K. and Randhawa, S.S. (2021). Clinical and haemato-biochemical observations in dogs naturally infected with canine monocytic ehrlichiosis. Exploratory Animal and Medical Research. 11(2): 214- 219.

    35. Smith, R.D., Ristic, M., Huxsoll, D.L. and Baylor, R.A. (1975). Platelet kinetics in canine ehrlichiosis. Infection and Immunity. 11(6): 1216-1221.

    36. Srikala, D., Satish Kumar, K., Amruth Kumar, V.V. and Tirumala Rao, D.S. (2012). Clinical and therapeutic aspects of canine monocytic ehrlichiosis. Indian Journal of Veterinary Medicine. 32(2): 109-110.

    37. Srivastava, M.K. and Srivastava, A. (2011). Canine ehrlichiosis in dog. Indian Journal of Veterinary Medicine. 31(2): 128- 129.

    38. Stich, R.W., Rikihisa, Y., Ewing, S.A., Needham, G.R., Grover, D.L. and Jittapalapong, S. (2002). Detection of Ehrlichia canis in canine carrier blood and in individual experimentally infected ticks with a p30-based PCR assay. Journal of Clinical Microbiology. 40(2): 540-546.

    39. Tamai, I.A. and Staji, H. (2025). Molecular identification and associated risk factors of Hepatozoon canis among dogs visiting veterinary clinics in Tehran, Iran. BMC Veterinary Research. 21(1): 660.

    40. Taylor, M.A., Coop, R.L. and Wall, R. (2015). Veterinary Parasitology. John Wiley and Sons, Chichester.

    41. Venugopal, V., Deepa, P.M., Muhasin Asaf, V.N. et al. (2026). Molecular epidemiology and phylogenetics of canine haemoprotozoan and rickettsial infections in southern India. Veterinary Parasitology: Regional Studies and Reports. 67: 1013 99.

    42. Waner, T. (2008). Hematopathological changes in dogs infected with Ehrlichia canis. Israel Journal of Veterinary Medicine. 63(1): 19.

    43. Waner, T., Harrus, S., Jongejan, F., Bark, H., Keysary, A. and Cornelissen, A.W. (2001). Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis. Veterinary Parasitology. 95(1): 1-15.

    44. Woody, B.J. and Hoskins, J.D. (1991). Ehrlichial diseases of dogs. Veterinary Clinics of North America: Small Animal Practice. 21: 75-98.
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)