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Studies on Correlation of Platelet Indices with Molecular Diagnosis of Ehrlichia canis and Babesia gibsoni in Dogs  

A
Avantika Sharma1,*
0000-0003-1116-8127
A
A.P.S. Brar2
0009-0000-9108-9650
H
Harkirat Singh2
0000-0003-1709-4480
1Department of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.
2Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.

ABSTRACT

Background: Though studies have been carried out globally regarding the correlation of platelet indices with canine monocytic ehrlichiosis and babesiosis, there appears to be limited reports on this aspect from India and there has been no published report from Punjab. The objective of this study is to correlate the role of platelet morphometry w.r.t size, colour, anisocytosis, polymorphism, activated or non- activated platelets and indices in the diagnosis of Ehrlichia canis and Babesia gibsoni infections in dogs.

Methods: A total of 500 suspected cases of dogs were analyzed. A questionnaire proforma containing information pertaining to various risk factors i.e., age, breed, gender, clinical history (presence of fever and ticks, epistaxis and respiratory distress/exercise intolerance) was prepared for investigation. The blood samples (2-3 mL) were collected in EDTA and serum vials for further examination.

Result: In dogs affected by Ehrlichia canis and Babesia gibsoni, platelet counts showed a marked reduction. Babesia-infected dogs exhibited a notable rise in mean platelet volume (MPV) and mean platelet mass (MPM), along with a decline in plateletcrit (PCT). Platelet distribution width (PDW) was elevated in both infections, while mean platelet component (MPC) increased in cases of severe thrombocytopenia associated with ehrlichiosis. In E. canis infection, a higher proportion of small, polychromic, non-activated platelets was observed, whereas B.gibsoni infection was characterized by larger, hypochromic, activated platelets. Both conditions also showed evidence of anisocytosis and polymorphism.

INTRODUCTION

Platelets constitute the primary defense mechanism against hemorrhage resulting from both microvascular and macrovascular injury. Disorders in platelet number or function commonly manifest as mucocutaneous bleeding (Hackner, 2009). Among companion animals, thrombocytopenia represents the most frequently encountered acquired hemostatic disorder, particularly in emergency veterinary practice. Multiple mechanisms underlie the development of thrombocytopenia, including sample dilution, accelerated clearance, impaired production, excessive consumption and splenic sequestration (Silverstein et al., 1985). In canine ehrlichiosis, a reduction in plateletcrit (PCT) has been documented (Moraes, 2001), whereas in babesiosis, decreased plateletcrit accompanied by an elevation in mean platelet volume (MPV) has been reported (Baranidharan et al., 2017).
       
Canine ehrlichiosis is an important and potentially fatal disease caused by an intracellular bacteria Ehrlichia canis, which parasitize monocytes and granulocytes and in chronic conditions is associated with irreversible bone marrow destruction (Skotarczak, 2003; Senthil and Chakravarthi, 2023). The disease is manifested by a wide variety of clinical and hematological signs and has been classified as acute, subclinical and chronic (Ristic and Holland, 1993).
       
Babesiosis is also one of the most important tick transmitted apicomplexan haemoprotozoan diseases that infect a wide range of vertebrate hosts and cause severe diseases in wild and domesticated animals (Kultler and Ristic, 1988). The most common and endemic strain of Babesia is Babesia gibsoni. The wide range of clinical symptoms, determines how severe babesiosis is, accompanied by signs like anorexia, lethargy, haemolytic anaemia, icterus, vomiting and marked loss of body condition (Ettinger and Feldman, 2005; Vial and Gorenflot, 2006; Choudhary et al., 2025).

MATERIALS AND METHODS

Platelet indices
 
A total of 500 suspected cases of dogs were analyzed. The platelet indices like Mean platelet volume (MPV), Plateletcrit (PCT), Platelet distribution width (PDW) and large platelet (LP) were recorded. Blood smears were stained by Leishman’s method. The platelet parameters were determined using ADVIA 2120 Hematology System and Mythic 18 Vet (Woodley Equipment Company Ltd. England) haematology analyser.
       
In addition, fifteen samples from apparently healthy animals presented for vaccination and routine health check up to the Centralized Clinical Diagnostic Laboratory in the Department of Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) Ludhiana (2021) were also analyzed and all the above parameters were recorded. The values were considered as control values for the comparison with dogs suffering from disease conditions.
 
Blood smear examination of platelets morphometry and counts
 
Peripheral smear examination was done to visualize platelet size, colour, polymorphism, anisocytosis,  presence of activated or non-activated platelets, measuring the length, width and area of the platelets in random 20 fields. The platelet count was done, by routine light microscopy of a Leishman’s stained peripheral smear Kopparthi et al., (2021). The count was done in oil immersion by multiplying the sum of the number of platelets counted in 8-10 fields less than 1000 x oil magnification by 2000. Every blood smear was carefully evaluated for the presence of platelet clumps that may falsely lower the platelet counts. The manual counts and the auto analyzer counts were correlated.
 
Molecular detection by duplex PCR
 
The assay protocol was standardized in the laboratory to amplify a 380-bp fragment of the E. canis virB9 gene was amplified by PCR using species-specific primers, as described by Kledmanee et al., (2009) and a 488-bp fragment of B. gibsoni 16S rRNA gene with published primers as outlined by Jain (1986) were included in the assay.
 
Statistical analysis
 
Analysis of the was done using suitable statistical software.

RESULTS AND DISCUSSION

History and clinical signs in dogs suffering from Ehrlichia canis infection
 
89 cases were of ehrlichiosis infected dogs, fever (53.68%) was the most predominant sign followed by anorexia (44.21%), melena (43.16%), weight loss (36.84%), epistaxis and presence of ticks (34.74%), vomition (30.53%), enlarged lymph nodes (27.37%), lethargy (25.26%), icteric mucus membrane (22.11%), nasal discharge (21.05%), corneal opacity (17.89%), anemia (14.74%), thrombocytopenia (13.68%) and dark coloured urine (11.58%). Dilipkumar et al., (2018) reported lymphadenopathy as the predominant clinical sign followed by pyrexia, depression, epistaxis, icteric mucus membrane, ascites and lameness. Ferreira  et al. (2018) reported epistaxis in 9.09% of the dogs positive for concomitant haemoparasitic infection. Likewise, Chatanga et al., (2021) reported 72.4% of the dogs affected with haemoparasitic infection had history of ticks along with anemia and leucopenia were the most significant hematological alteration (Gamit et al., 2024).
 
History and clinical signs in dogs suffering from Babesia gibsoni
 
Of the 68 cases of babesiosis, vomition (59.46%) was the most predominant sign, followed by diarrhea (44.59%), fever (40.54%), ticks (33.78%), inappetence (32.43%), abdominal distension and hematuria (28.38%), icteric mucus membrane (25.68%), lethargy (22.97%) melena (20.27%) and jaundice (12.16%). Tufani (2014); Gonde et al., (2017); Parvathy et al., (2019) reported weakness, anorexia, pale mucus membrane, fever and vomition. Peters et al., (2005) reported dogs with renal failure showed vomition as GIT sign associated with uremic gastritis.
 
Association of physical examination of dogs with ehrlichiosis and babesiosis
 
The relationship between physical examination findings and parasitic infections in dogs was evaluated. Overall, no statistically significant association was observed between clinical examination outcomes and the presence of babesiosis or ehrlichiosis. However, certain trends were noted: a dull appearance was more frequently recorded in dogs with ehrlichiosis (64.29%) compared to those with babesiosis (35.71%). Similarly, pale mucous membranes were more prevalent in ehrlichiosis (64.29%) than in babesiosis (35.71%). In contrast, congested mucous membranes were more commonly observed in babesiosis (61.11%) relative to ehrlichiosis (38.89%). Lymphadenopathy was also more frequent in ehrlichiosis cases (65%) than in babesiosis (35%). Elevated body temperature (mean 103.8°F) was consistently observed in dogs infected with either parasite. Comparable findings have been reported in the literature: Yadav (2022) described hemoprotozoan-infected dogs as dull, depressed and exhibiting pale mucous membranes with lymphadenopathy, while Ghosh et al., (2020) documented pyrexia in both ehrlichiosis and babesiosis. Furthermore, Ferreira et al., (2018) noted that 68.18% of dogs with concurrent tick-borne infections presented with fever.
 
Study of platelet morphometry in dogs having ehrlichiosis and babesiosis
 
The platelets morphology was studied w.r.t staining characteristic, size, polymorphism, anisocytosis, activated and non-activated platelets. The cases of dogs having ehrlichiosis showed that the value of platelet length varied from 1.62-4.03 (µm) with a mean value of 2.43 (µm) which was within the normal range. The value of platelet width varied from 1.02-2.77(µm) with a mean value of 1.69 (µm) which was within the normal range. The value of platelet area varied from 2.01-9.87 (µm2) with a mean value of 4.06 (µm2) which was within the normal range. Whereas, babesiosis affected dogs showed that the value of platelet length varied from 1.56-3.95 (µm) with a mean value of 2.99 (µm) which was within the normal range. The value of platelet width varied from 1.05-2.72 (µm) with a mean value of 1.97 (µm) which was within the normal range. The value of platelet area varied from 1.44-9.78 (µm2) with a mean value of 5.72 (µm2) which was within the normal range. Rebar et al., (2001) reported that in a healthy dog, platelets in general measure 2.2-3.7 microns in diameter and 0.5 microns thick and circulate at a concentration of approximately 200, 000/µl to 600, 000/µl. Stobbl, (1959) reported that morphologic features of thrombocytes were altered by a variety of inherited and acquired conditions. Bessis (1972) reported that megathrombocytes as a sign of ineffective thrombopoiesis or increased demands for platelets at periphery. The percentage of mega thrombocytes (size of platelets was ≥5 µm as measured by an eyepiece micrometer) present was estimated following serial evaluation of 100 platelets (x 1000 magnification) in buffy-coat smears as described by Mylonakis et al., (2004).
 
The prevalence of anisocytosis and polymorphism of platelets in dogs having ehrlichiosis and babesiosis
 
The prevalence of anisocytosis and polymorphism of platelets in dogs having ehrlichiosis and babesiosis was studied. Anisocytosis and polymorphism (Fig 1) was observed in ehrlichiosis and babesiosis affected dogs with (55%) and (42%) frequency. Bessis (1972) reported anisocytosis as the sign of disturbed thrombopoiesis and similar to our study, Zygner et al., (2007) also reported anisocytosis in dogs with babesiosis.

Fig 1: Peripheral blood smear revealing polymorphism of platelet. Leishman stain x 100X.


 
Association of platelets morphometry in E. canis and B. gibsoni affected dogs on the basis of activated or non- activated platelets
 
Overall, there was a significant association between the activation of the platelets of dogs with ehrlichiosis and babesiosis (U«2 46.78; p<0.01). The proportion of cases with activated platelets were significantly higher in dogs with babesiosis (80.9%) as compared to dogs with ehrlichiosis (25.84%) (Fig 2). On the other side, the proportion of cases with non-activated platelets were significantly higher in dogs having ehrlichiosis (74.16%) as compared to dogs having babesiosis (19.12%). With accordance to our study, Halmey et al., (2005) reported that in babesiosis, thick cytoplasmic processes with well-known fine pseudopods were found on the surface of platelets. Preena et al., (2021) also reported the presence of large activated platelets or macro-platelets in dogs with babesiosis.

Fig 2: Peripheral blood smear revealing activated platelet. Leishman stain x 100X.


 
Association of platelets morphometry in E. canis and B. gibsoni affected dogs on the basis of intensity of platelets colour
 
Overall, there was a significant association between the colour of the platelets of dogs with babesiosis and ehrlichiosis (c2 71.95; p<0.01). The proportion of cases with normochromic platelets were significantly higher in dogs having babesiosis (7.35%) as compared to dogs having ehrlichiosis (6.74%). The proportion of cases with hypochromic platelets were significantly higher in dogs having babesiosis (72.06%) as compared to dogs having ehrlichiosis (28.09%) (Fig 3). The proportion of cases with polychromic platelets were significantly higher in dogs having ehrlichiosis (65.17%) as compared to dogs having babesiosis (20.6%) (Fig 4). Halmay et al., (2005) reported that the alterations found in platelet morphology were not specific for any disorder and the most common platelet abnormalities observed were polychromasia.

Fig 3: Peripheral blood smear revealing hypochromic platelet. Leishman stain x 100X.



Fig 4: Peripheral blood smear revealing polychromic platelet. Leishman stain x 100X.


 
Comparison of platelet morphometry in E. canis and B. gibsoni dogs with apparently healthy dogs
 
The mean value of length in dogs suffering from babesiosis was significantly higher than the dogs having ehrlichiosis. The mean value of width in dogs was significantly higher in dogs having babesiosis than the dogs having ehrlichiosis. The mean value of area was significantly higher in dogs with babesiosis than the dogs with ehrlichiosis. (p<0.01). Halmay et al., (2005) reported that the alterations found in platelet morphology were not specific for any disorder and the most common platelet abnormalities observed were the presence of giant platelets which occurred in a high number in disorders accompanied by bleeding or haemolysis.

Comparisons of platelet indices in E. canis and B. gibsoni dogs with apparently healthy dogs
 
The comparisons of platelet indices in E. canis and B. gibsoni dogs with apparently healthy dogs was investigated. The mean value of MPV was significantly higher in dogs suffering from babesiosis than ehrlichiosis as compared to apparently healthy dogs (p<0.001). The mean value of PCT was significantly lower in dogs having ehrlichiosis and babesiosis as compared to apparently healthy dogs (p 0.04). The mean value of PDW was non- significantly higher in dogs having ehrlichiosis and babesiosis as compared to apparently healthy dogs (p 0.092). The mean value of MPC was non- significantly higher in dogs having ehrlichiosis as compared to apparently healthy dogs (p 0.155). The mean value of MPM was significantly higher in dogs having ehrlichiosis and babesiosis as compared to apparently healthy dogs (p <0.01). The mean value of large platelet was non-significantly higher in dogs having babesiosis and lower in dogs having ehrlichiosis as compared to apparently healthy dogs (p 0.08) (Table 1). In accordance, Moraes (2001) reported drop in PCT in dogs with ehrlichiosis which can be elucidated on the basis of thrombocytopenia. whereas, Kumar et al., (2021) reported increase in the PCT values in ehrlichia affected dogs as compared to control group. Zvorc et al. (2010); Kettner et al., (2003); Baranidharan  et al. (2017) reported decreased PCT and increased MPV in babesiosis affected dogs. Preena et al., (2021) also reported increased MPV, MPM and decreased MPC in canine babesiosis.

Table 1: Comparison of platelet indices in E. canis, B. gibsoni dogs with apparently healthy dogs (Mean0.08S.E).


 
Platelet indices in ehrlichiosis induced mild, moderate and severe thrombocytopenic dogs
 
The mean platelet counts, thrombocytopenia divided into mild thrombocytopenia (50,000-100,000 cells/cmm), moderate thrombocytopenia (25,000-50,000 cells/cmm) and severe thrombocytopenia (less than 25,000 cells/cmm) and their platelet indices including MPV, PCT, PDW, MPC, MPM and Large platelet (LP) in ehrlichiosis induced thrombocytopenia were analyzed and tabulated. The PCT showed a marked significant difference in between the control and the three groups of mild, moderate and severe thrombocytopenia whereas, MPV, PDW, MPC, MPM and LP did not show any significant difference among the three groups. Similarly, Breitschwerdt (2000) recorded the lower platelet counts in ehrlichiosis whereas Baranidharan et al., (2017) reported that PCT showed a marked significant difference in between the control and the three groups of mild, moderate and severe thrombocytopenia, however, MPV and PDW did not showed any significant difference among the three groups.
 
Platelet indices in babesiosis induced mild, moderate and severe thrombocytopenic dogs
 
The mean platelet counts and their platelet indices including MPV, PCT, PDW, MPC, MPM and Large platelet (LP) in babesiosis induced thrombocytopenia were analyzed and tabulated. As compared to control and the three groups of mild, moderate and severe thrombocytopenia, the PCT and PDW showed a marked significant difference whereas MPV, MPC, MPM and LP did not show any significant difference among the three groups. However, the MPC showed increased levels in the severe thrombocytopenia. Similarly, Breitschwerdt, (2000) recorded the lower platelet counts in babesiosis. Baranidharan et al., (2017) observed that PCT showed a marked significant difference whereas the PDW did not had any significant difference between the three groups, whereas, the MPV showed increased levels in the severe TCP group of babesiosis. In the severe thrombocytopenia, there was significant decrease in the platelet count of both E. canis and B. gibsoni affected dogs and the decrease was more appreciable in ehrlichiosis.
       
The study on correlation of platelet indices with molecular diagnosis of Ehrlichia canis and Babesia gibsoni is very useful for field veterinarians and local pet owners. It helps in early suspicion of these infections using simple blood tests, even when advanced molecular tests are not easily available. Platelet changes can guide timely treatment and reduce disease severity. This lowers treatment cost and improves recovery of dogs. The study also promotes awareness about tick-borne diseases and tick control. Thus, it supports better disease management at the field level.

CONCLUSION

The present study highlights that platelet indices and morphometric alterations can provide important supportive diagnostic information in canine infections caused by Ehrlichia canis and Babesia gibsoni. Both infections were associated with marked thrombocytopenia and significant variations in platelet parameters. Dogs infected with B. gibsoni showed increased MPV, MPM and PDW with decreased PCT, indicating the presence of larger and activated platelets. In contrast, E. canis infection exhibited a higher proportion of small, polychromic, non-activated platelets with elevated MPC in severe thrombocytopenia. Morphological changes such as anisocytosis and polymorphism were evident in both infections. These findings suggest that platelet morphometry and indices can aid in the early diagnosis and differentiation of these haemoprotozoan infections in dogs.
 

ACKNOWLEDGEMENT

The authors express their sincere gratitude to the Department of Veterinary Pathology and Department of Veterinary Parasitology, College of Veterinary Science and Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, for providing the necessary infrastructure and facilities to carry out this research. We extend our heartfelt thanks to the laboratory staff and technical assistants for their valuable help during sample collection and processing. We acknowledge the contributions of our mentors, colleagues and well-wishers whose guidance and encouragement helped shape this work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal Care and Handling Techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Studies on Correlation of Platelet Indices with Molecular Diagnosis of Ehrlichia canis and Babesia gibsoni in Dogs  

    A
    Avantika Sharma1,*
    0000-0003-1116-8127
    A
    A.P.S. Brar2
    0009-0000-9108-9650
    H
    Harkirat Singh2
    0000-0003-1709-4480
    1Department of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.
    2Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.

    ABSTRACT

    Background: Though studies have been carried out globally regarding the correlation of platelet indices with canine monocytic ehrlichiosis and babesiosis, there appears to be limited reports on this aspect from India and there has been no published report from Punjab. The objective of this study is to correlate the role of platelet morphometry w.r.t size, colour, anisocytosis, polymorphism, activated or non- activated platelets and indices in the diagnosis of Ehrlichia canis and Babesia gibsoni infections in dogs.

    Methods: A total of 500 suspected cases of dogs were analyzed. A questionnaire proforma containing information pertaining to various risk factors i.e., age, breed, gender, clinical history (presence of fever and ticks, epistaxis and respiratory distress/exercise intolerance) was prepared for investigation. The blood samples (2-3 mL) were collected in EDTA and serum vials for further examination.

    Result: In dogs affected by Ehrlichia canis and Babesia gibsoni, platelet counts showed a marked reduction. Babesia-infected dogs exhibited a notable rise in mean platelet volume (MPV) and mean platelet mass (MPM), along with a decline in plateletcrit (PCT). Platelet distribution width (PDW) was elevated in both infections, while mean platelet component (MPC) increased in cases of severe thrombocytopenia associated with ehrlichiosis. In E. canis infection, a higher proportion of small, polychromic, non-activated platelets was observed, whereas B.gibsoni infection was characterized by larger, hypochromic, activated platelets. Both conditions also showed evidence of anisocytosis and polymorphism.

    INTRODUCTION

    Platelets constitute the primary defense mechanism against hemorrhage resulting from both microvascular and macrovascular injury. Disorders in platelet number or function commonly manifest as mucocutaneous bleeding (Hackner, 2009). Among companion animals, thrombocytopenia represents the most frequently encountered acquired hemostatic disorder, particularly in emergency veterinary practice. Multiple mechanisms underlie the development of thrombocytopenia, including sample dilution, accelerated clearance, impaired production, excessive consumption and splenic sequestration (Silverstein et al., 1985). In canine ehrlichiosis, a reduction in plateletcrit (PCT) has been documented (Moraes, 2001), whereas in babesiosis, decreased plateletcrit accompanied by an elevation in mean platelet volume (MPV) has been reported (Baranidharan et al., 2017).
           
    Canine ehrlichiosis is an important and potentially fatal disease caused by an intracellular bacteria Ehrlichia canis, which parasitize monocytes and granulocytes and in chronic conditions is associated with irreversible bone marrow destruction (Skotarczak, 2003; Senthil and Chakravarthi, 2023). The disease is manifested by a wide variety of clinical and hematological signs and has been classified as acute, subclinical and chronic (Ristic and Holland, 1993).
           
    Babesiosis is also one of the most important tick transmitted apicomplexan haemoprotozoan diseases that infect a wide range of vertebrate hosts and cause severe diseases in wild and domesticated animals (Kultler and Ristic, 1988). The most common and endemic strain of Babesia is Babesia gibsoni. The wide range of clinical symptoms, determines how severe babesiosis is, accompanied by signs like anorexia, lethargy, haemolytic anaemia, icterus, vomiting and marked loss of body condition (Ettinger and Feldman, 2005; Vial and Gorenflot, 2006; Choudhary et al., 2025).

    MATERIALS AND METHODS

    Platelet indices
     
    A total of 500 suspected cases of dogs were analyzed. The platelet indices like Mean platelet volume (MPV), Plateletcrit (PCT), Platelet distribution width (PDW) and large platelet (LP) were recorded. Blood smears were stained by Leishman’s method. The platelet parameters were determined using ADVIA 2120 Hematology System and Mythic 18 Vet (Woodley Equipment Company Ltd. England) haematology analyser.
           
    In addition, fifteen samples from apparently healthy animals presented for vaccination and routine health check up to the Centralized Clinical Diagnostic Laboratory in the Department of Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) Ludhiana (2021) were also analyzed and all the above parameters were recorded. The values were considered as control values for the comparison with dogs suffering from disease conditions.
     
    Blood smear examination of platelets morphometry and counts
     
    Peripheral smear examination was done to visualize platelet size, colour, polymorphism, anisocytosis,  presence of activated or non-activated platelets, measuring the length, width and area of the platelets in random 20 fields. The platelet count was done, by routine light microscopy of a Leishman’s stained peripheral smear Kopparthi et al., (2021). The count was done in oil immersion by multiplying the sum of the number of platelets counted in 8-10 fields less than 1000 x oil magnification by 2000. Every blood smear was carefully evaluated for the presence of platelet clumps that may falsely lower the platelet counts. The manual counts and the auto analyzer counts were correlated.
     
    Molecular detection by duplex PCR
     
    The assay protocol was standardized in the laboratory to amplify a 380-bp fragment of the E. canis virB9 gene was amplified by PCR using species-specific primers, as described by Kledmanee et al., (2009) and a 488-bp fragment of B. gibsoni 16S rRNA gene with published primers as outlined by Jain (1986) were included in the assay.
     
    Statistical analysis
     
    Analysis of the was done using suitable statistical software.

    RESULTS AND DISCUSSION

    History and clinical signs in dogs suffering from Ehrlichia canis infection
     
    89 cases were of ehrlichiosis infected dogs, fever (53.68%) was the most predominant sign followed by anorexia (44.21%), melena (43.16%), weight loss (36.84%), epistaxis and presence of ticks (34.74%), vomition (30.53%), enlarged lymph nodes (27.37%), lethargy (25.26%), icteric mucus membrane (22.11%), nasal discharge (21.05%), corneal opacity (17.89%), anemia (14.74%), thrombocytopenia (13.68%) and dark coloured urine (11.58%). Dilipkumar et al., (2018) reported lymphadenopathy as the predominant clinical sign followed by pyrexia, depression, epistaxis, icteric mucus membrane, ascites and lameness. Ferreira  et al. (2018) reported epistaxis in 9.09% of the dogs positive for concomitant haemoparasitic infection. Likewise, Chatanga et al., (2021) reported 72.4% of the dogs affected with haemoparasitic infection had history of ticks along with anemia and leucopenia were the most significant hematological alteration (Gamit et al., 2024).
     
    History and clinical signs in dogs suffering from Babesia gibsoni
     
    Of the 68 cases of babesiosis, vomition (59.46%) was the most predominant sign, followed by diarrhea (44.59%), fever (40.54%), ticks (33.78%), inappetence (32.43%), abdominal distension and hematuria (28.38%), icteric mucus membrane (25.68%), lethargy (22.97%) melena (20.27%) and jaundice (12.16%). Tufani (2014); Gonde et al., (2017); Parvathy et al., (2019) reported weakness, anorexia, pale mucus membrane, fever and vomition. Peters et al., (2005) reported dogs with renal failure showed vomition as GIT sign associated with uremic gastritis.
     
    Association of physical examination of dogs with ehrlichiosis and babesiosis
     
    The relationship between physical examination findings and parasitic infections in dogs was evaluated. Overall, no statistically significant association was observed between clinical examination outcomes and the presence of babesiosis or ehrlichiosis. However, certain trends were noted: a dull appearance was more frequently recorded in dogs with ehrlichiosis (64.29%) compared to those with babesiosis (35.71%). Similarly, pale mucous membranes were more prevalent in ehrlichiosis (64.29%) than in babesiosis (35.71%). In contrast, congested mucous membranes were more commonly observed in babesiosis (61.11%) relative to ehrlichiosis (38.89%). Lymphadenopathy was also more frequent in ehrlichiosis cases (65%) than in babesiosis (35%). Elevated body temperature (mean 103.8°F) was consistently observed in dogs infected with either parasite. Comparable findings have been reported in the literature: Yadav (2022) described hemoprotozoan-infected dogs as dull, depressed and exhibiting pale mucous membranes with lymphadenopathy, while Ghosh et al., (2020) documented pyrexia in both ehrlichiosis and babesiosis. Furthermore, Ferreira et al., (2018) noted that 68.18% of dogs with concurrent tick-borne infections presented with fever.
     
    Study of platelet morphometry in dogs having ehrlichiosis and babesiosis
     
    The platelets morphology was studied w.r.t staining characteristic, size, polymorphism, anisocytosis, activated and non-activated platelets. The cases of dogs having ehrlichiosis showed that the value of platelet length varied from 1.62-4.03 (µm) with a mean value of 2.43 (µm) which was within the normal range. The value of platelet width varied from 1.02-2.77(µm) with a mean value of 1.69 (µm) which was within the normal range. The value of platelet area varied from 2.01-9.87 (µm2) with a mean value of 4.06 (µm2) which was within the normal range. Whereas, babesiosis affected dogs showed that the value of platelet length varied from 1.56-3.95 (µm) with a mean value of 2.99 (µm) which was within the normal range. The value of platelet width varied from 1.05-2.72 (µm) with a mean value of 1.97 (µm) which was within the normal range. The value of platelet area varied from 1.44-9.78 (µm2) with a mean value of 5.72 (µm2) which was within the normal range. Rebar et al., (2001) reported that in a healthy dog, platelets in general measure 2.2-3.7 microns in diameter and 0.5 microns thick and circulate at a concentration of approximately 200, 000/µl to 600, 000/µl. Stobbl, (1959) reported that morphologic features of thrombocytes were altered by a variety of inherited and acquired conditions. Bessis (1972) reported that megathrombocytes as a sign of ineffective thrombopoiesis or increased demands for platelets at periphery. The percentage of mega thrombocytes (size of platelets was ≥5 µm as measured by an eyepiece micrometer) present was estimated following serial evaluation of 100 platelets (x 1000 magnification) in buffy-coat smears as described by Mylonakis et al., (2004).
     
    The prevalence of anisocytosis and polymorphism of platelets in dogs having ehrlichiosis and babesiosis
     
    The prevalence of anisocytosis and polymorphism of platelets in dogs having ehrlichiosis and babesiosis was studied. Anisocytosis and polymorphism (Fig 1) was observed in ehrlichiosis and babesiosis affected dogs with (55%) and (42%) frequency. Bessis (1972) reported anisocytosis as the sign of disturbed thrombopoiesis and similar to our study, Zygner et al., (2007) also reported anisocytosis in dogs with babesiosis.

    Fig 1: Peripheral blood smear revealing polymorphism of platelet. Leishman stain x 100X.


     
    Association of platelets morphometry in E. canis and B. gibsoni affected dogs on the basis of activated or non- activated platelets
     
    Overall, there was a significant association between the activation of the platelets of dogs with ehrlichiosis and babesiosis (U«2 46.78; p<0.01). The proportion of cases with activated platelets were significantly higher in dogs with babesiosis (80.9%) as compared to dogs with ehrlichiosis (25.84%) (Fig 2). On the other side, the proportion of cases with non-activated platelets were significantly higher in dogs having ehrlichiosis (74.16%) as compared to dogs having babesiosis (19.12%). With accordance to our study, Halmey et al., (2005) reported that in babesiosis, thick cytoplasmic processes with well-known fine pseudopods were found on the surface of platelets. Preena et al., (2021) also reported the presence of large activated platelets or macro-platelets in dogs with babesiosis.

    Fig 2: Peripheral blood smear revealing activated platelet. Leishman stain x 100X.


     
    Association of platelets morphometry in E. canis and B. gibsoni affected dogs on the basis of intensity of platelets colour
     
    Overall, there was a significant association between the colour of the platelets of dogs with babesiosis and ehrlichiosis (c2 71.95; p<0.01). The proportion of cases with normochromic platelets were significantly higher in dogs having babesiosis (7.35%) as compared to dogs having ehrlichiosis (6.74%). The proportion of cases with hypochromic platelets were significantly higher in dogs having babesiosis (72.06%) as compared to dogs having ehrlichiosis (28.09%) (Fig 3). The proportion of cases with polychromic platelets were significantly higher in dogs having ehrlichiosis (65.17%) as compared to dogs having babesiosis (20.6%) (Fig 4). Halmay et al., (2005) reported that the alterations found in platelet morphology were not specific for any disorder and the most common platelet abnormalities observed were polychromasia.

    Fig 3: Peripheral blood smear revealing hypochromic platelet. Leishman stain x 100X.



    Fig 4: Peripheral blood smear revealing polychromic platelet. Leishman stain x 100X.


     
    Comparison of platelet morphometry in E. canis and B. gibsoni dogs with apparently healthy dogs
     
    The mean value of length in dogs suffering from babesiosis was significantly higher than the dogs having ehrlichiosis. The mean value of width in dogs was significantly higher in dogs having babesiosis than the dogs having ehrlichiosis. The mean value of area was significantly higher in dogs with babesiosis than the dogs with ehrlichiosis. (p<0.01). Halmay et al., (2005) reported that the alterations found in platelet morphology were not specific for any disorder and the most common platelet abnormalities observed were the presence of giant platelets which occurred in a high number in disorders accompanied by bleeding or haemolysis.

    Comparisons of platelet indices in E. canis and B. gibsoni dogs with apparently healthy dogs
     
    The comparisons of platelet indices in E. canis and B. gibsoni dogs with apparently healthy dogs was investigated. The mean value of MPV was significantly higher in dogs suffering from babesiosis than ehrlichiosis as compared to apparently healthy dogs (p<0.001). The mean value of PCT was significantly lower in dogs having ehrlichiosis and babesiosis as compared to apparently healthy dogs (p 0.04). The mean value of PDW was non- significantly higher in dogs having ehrlichiosis and babesiosis as compared to apparently healthy dogs (p 0.092). The mean value of MPC was non- significantly higher in dogs having ehrlichiosis as compared to apparently healthy dogs (p 0.155). The mean value of MPM was significantly higher in dogs having ehrlichiosis and babesiosis as compared to apparently healthy dogs (p <0.01). The mean value of large platelet was non-significantly higher in dogs having babesiosis and lower in dogs having ehrlichiosis as compared to apparently healthy dogs (p 0.08) (Table 1). In accordance, Moraes (2001) reported drop in PCT in dogs with ehrlichiosis which can be elucidated on the basis of thrombocytopenia. whereas, Kumar et al., (2021) reported increase in the PCT values in ehrlichia affected dogs as compared to control group. Zvorc et al. (2010); Kettner et al., (2003); Baranidharan  et al. (2017) reported decreased PCT and increased MPV in babesiosis affected dogs. Preena et al., (2021) also reported increased MPV, MPM and decreased MPC in canine babesiosis.

    Table 1: Comparison of platelet indices in E. canis, B. gibsoni dogs with apparently healthy dogs (Mean0.08S.E).


     
    Platelet indices in ehrlichiosis induced mild, moderate and severe thrombocytopenic dogs
     
    The mean platelet counts, thrombocytopenia divided into mild thrombocytopenia (50,000-100,000 cells/cmm), moderate thrombocytopenia (25,000-50,000 cells/cmm) and severe thrombocytopenia (less than 25,000 cells/cmm) and their platelet indices including MPV, PCT, PDW, MPC, MPM and Large platelet (LP) in ehrlichiosis induced thrombocytopenia were analyzed and tabulated. The PCT showed a marked significant difference in between the control and the three groups of mild, moderate and severe thrombocytopenia whereas, MPV, PDW, MPC, MPM and LP did not show any significant difference among the three groups. Similarly, Breitschwerdt (2000) recorded the lower platelet counts in ehrlichiosis whereas Baranidharan et al., (2017) reported that PCT showed a marked significant difference in between the control and the three groups of mild, moderate and severe thrombocytopenia, however, MPV and PDW did not showed any significant difference among the three groups.
     
    Platelet indices in babesiosis induced mild, moderate and severe thrombocytopenic dogs
     
    The mean platelet counts and their platelet indices including MPV, PCT, PDW, MPC, MPM and Large platelet (LP) in babesiosis induced thrombocytopenia were analyzed and tabulated. As compared to control and the three groups of mild, moderate and severe thrombocytopenia, the PCT and PDW showed a marked significant difference whereas MPV, MPC, MPM and LP did not show any significant difference among the three groups. However, the MPC showed increased levels in the severe thrombocytopenia. Similarly, Breitschwerdt, (2000) recorded the lower platelet counts in babesiosis. Baranidharan et al., (2017) observed that PCT showed a marked significant difference whereas the PDW did not had any significant difference between the three groups, whereas, the MPV showed increased levels in the severe TCP group of babesiosis. In the severe thrombocytopenia, there was significant decrease in the platelet count of both E. canis and B. gibsoni affected dogs and the decrease was more appreciable in ehrlichiosis.
           
    The study on correlation of platelet indices with molecular diagnosis of Ehrlichia canis and Babesia gibsoni is very useful for field veterinarians and local pet owners. It helps in early suspicion of these infections using simple blood tests, even when advanced molecular tests are not easily available. Platelet changes can guide timely treatment and reduce disease severity. This lowers treatment cost and improves recovery of dogs. The study also promotes awareness about tick-borne diseases and tick control. Thus, it supports better disease management at the field level.

    CONCLUSION

    The present study highlights that platelet indices and morphometric alterations can provide important supportive diagnostic information in canine infections caused by Ehrlichia canis and Babesia gibsoni. Both infections were associated with marked thrombocytopenia and significant variations in platelet parameters. Dogs infected with B. gibsoni showed increased MPV, MPM and PDW with decreased PCT, indicating the presence of larger and activated platelets. In contrast, E. canis infection exhibited a higher proportion of small, polychromic, non-activated platelets with elevated MPC in severe thrombocytopenia. Morphological changes such as anisocytosis and polymorphism were evident in both infections. These findings suggest that platelet morphometry and indices can aid in the early diagnosis and differentiation of these haemoprotozoan infections in dogs.
     

    ACKNOWLEDGEMENT

    The authors express their sincere gratitude to the Department of Veterinary Pathology and Department of Veterinary Parasitology, College of Veterinary Science and Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, for providing the necessary infrastructure and facilities to carry out this research. We extend our heartfelt thanks to the laboratory staff and technical assistants for their valuable help during sample collection and processing. We acknowledge the contributions of our mentors, colleagues and well-wishers whose guidance and encouragement helped shape this work.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal Care and Handling Techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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