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Full Research Article

Prevalence of Ehrlichiosis in Dogs with Gastrointestinal Bleeding Signs and Meta-analysis of Causal Association

S
Saravanan Subramanian1,*
K
Kathirvel Shanmugam2
S
Sumathi Devarajan3
D
Dhivya Praba Ramasamy Abimanyu1
S
Selvaraju Ganapathy1
1Department of Veterinary Public Health and Epidemiology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.
2Department of Veterinary Surgery, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.
3Department of Veterinary Clinical Medicine, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.

ABSTRACT

Background: Most of the canine monocytic ehrlichiosis (CME) cases are left undetected until they reach the chronic stage which is non-responsive to the treatment resulting in death associated with panleucopenia. Hence, diagnosis of clinical or subclinical CME is of paramount importance to prevent entering into the fatal chronic stage.

Methods: Dogs (n=50) presented with haemorrhagic signs were subjected to microscopic examination and immunochromatography test. Positive cases were subjected to haemato-biochemical analysis, radiography, ultrasonography and computerized tomography. Meta-analysis of clinical variables was done to determine the causal association.

Result: CME was observed in 22.0 per cent of cases by lateral flow assay (LFA) and 2.0 per cent by microscopic examination. Pathological changes were noticed in the liver, spleen, kidney, lungs, urinary bladder and intestine. A decrease (P<0.05) in haemoglobin level (8.66±1.59), increase (p<0.05) in white blood cell (21.87±5.2), monocyte count (8.5±1.06) and serum alkaline phosphatase level (151.33±48.12) and increase (p<0.01) in blood urea nitrogen (57.64±12.82) was observed. Melena (91.6%) was the predominant sign among positive cases. The sign- specific prevalence rate was high for epistaxis, circling/ joint pain (100.0%), emaciation (100.0%) and monocytosis (75.0%). The relative risk for the prevalence of CME was 5 to 6 times more in dogs with emaciation (95% CI, 2.26 to 12.18) and melena (95% CI, 0.73 to 6.71). High prevalence was observed in females (28.5%, p>0.05), 4-6-year-old (45.0%, p>0.05), pure breeds (100.0%, p<0.01) and recent tick infestation (31.2%, p>0.05). Antibody-based test, imaging techniques, haemato-biochemical analysis and the causal association could be useful tools in identifying the persistent ehrlichial infection.

INTRODUCTION

Canine monocytic ehrlichiosis (CME) is a significant vector borne disease caused by the obligate intracellular bacteria, Ehrlichia canis, transmitted by the tick, Rhipicephalus sanguineus and dogs act as reservoir (Aziz et al., 2022). The pathogen attacks macrophages and leucocytes in dogs (Mylonakis and Theodorou, 2017) and is reported to be zoonotic (Andric, 2014).
       
The CME occurs as acute, subacute and chronic, with the clinical findings such as fever, lethargy, anaemia, lymphadenomegaly, hepatomegaly, petechiae or ecchymoses, vasculitis and extended bleeding period during estrus (Aziz et al., 2022). Some dogs due to ineffective treatment may become subclinical carriers for months or years by evasion of immune response (Greene at al., 2012). Most of dogs do not show any visible signs except mild thrombocytopenia (Maggi et al., 2014) and may enter into the most fatal chronic stage (myelosuppressive form). In chronic form, bleeding disorders such as ecchymoses, epistaxis, haematuria, melena and prolonged bleeding from venipuncture sites are more common (Mylonakis et al., 2019).
       
The diagnosis of the ehrlichiosis becomes challenging owing to the several overlapping and non-specific clinical signs (Bai et al., 2017). Further, in recovered cases, the low ehrlichaemia is undetectable by molecular methods such as polymerase chain reaction (PCR) (Theodorou et al., 2013).  An early diagnosis would help in prompt treatment of cases of ehrlichiosis before entering into the fatal chronic stage (Aziz et al., 2022). Hence, the study is undertaken to report the early diagnosis of CME by lateral flow assay, to identify changes in the organs and to determine the causal association in the occurrence of CME.

MATERIALS AND METHODS

A cross-sectional study in the dogs (n=50) presented with the signs of gastrointestinal bleeding in 2023 to the Veterinary Clinical Complex, Veterinary College and Research Institute, Namakkal was conducted. Serum samples were subjected to lateral flow immuno assay based immunochromatography test (Pet X®, J and B Biotech Ltd, UK) for detection of E. canis antibodies. The positive result was indicated by the red lines both in control (C) and test (T) zones, whereas, negative result was indicated by the red line only in C zone. Giemsa stained peripheral blood smears were subjected to microscopic examination.
       
The prevalence rate was statistically analysed between groups by Chi-square test. Blood were collected for haemato-biochemical analysis by auto Veterinary hematology Analyzer (Rayto®, Version 2.4e, RT7600Vet, Rayto Life and Analytical Sciences Co., Ltd., China) and auto Biochemistry analyzer (A15 Biosystems SA, Spain and the values were compared with healthy control group by Student t-test (using SPSS software). Radiography, ultrasonography (USG) (Esoate®, Italy) and computerized tomography (CT) (Toshiba Alexion 16 Slice CT scanner, Japan) were performed in positive cases. The causal association for the clinical variables was determined by logistic regression analysis using Med calc Statistical software and a forest tree analysis of RR (CI:95%) was plotted on a logarithmic scale.

RESULTS AND DISCUSSION

Microscopic examination (× 100) could detect morulae of ehrlichia within monocytes (Fig 1) in one case (2.0 per cent), whereas sandwich lateral flow assay could detect positivity (Fig 1) in 11 cases (22.0 per cent) and an overall prevalence of 24.0 per cent was observed for CME in dogs. Kappa analysis revealed only a slight agreement (Kappa value=0.18) between the tests (Landis and Koch, 1977). Ehrlichia could not be detected in most of the cases owing to the poor sensitivity of microscopic examination (Singh et al., 2021; Angkanaporn et al., 2022), as detection of morulae could be possible only in 4 to 6% of cases (Aziz et al., 2022; Sarawade et al., 2023; Gamit et al., 2024 and Checa et al., 2024). Previously, Senthil et al. (2023) recorded a highest prevalence of 56.37 per cent of E. canis in dogs from Chennai city. In this study, the immunochromatography test could detect more ehrlichia infections before entering into chronic form. Serological tests could detect both current and past ehrlichia infections and molecular test is recommended to distinguish the active infection (Sainz et al., 2015). Immuno chromato- graphy test (ICT) is reported to be a quick and useful diagnostic tool with 87% sensitivity and 95% specificity (Geromichalou and Faixova, 2017). Previously, Checa et al. (2024) also reported a high prevalence of CME by LFA.

Fig 1: (a) Microscopic examination of Giemsa stained blood smears showing morulae of E. canis within monocyte (´ 100); (b)Lateral flow assay based immunochromatography test showing positivity for CME and (c) LFA showing negativity for CME.


       
The clinical manifestations of positive cases are presented in Table 1 and Fig 2. From Table 1, most of the cases (84.4%) were found to be sub-acute as they showed clinical signs without fever (Waner and Harrus, 2013). Melena, anaemia, lymphadenopathy and thrombocytopenia were the most common signs observed in the positive cases which concurred to the findings of Singh et al. (2021). The bleeding signs could be associated with anti-platelet antibodies or bone marrow suppression or vasculitis (Asawapattanakul et al., 2021; Aziz et al., 2022). A very few were chronic cases (16.6%) showing panleucopenia, as chronic CME is indicated by severe pancytopenia due to myelosuppression caused by the ehrlichiae (Sainz et al., 2015; Singh et al. 2021). 

Table 1: Distribution of signs in the positive cases of CME with gastrointestinal bleeding signs.



Fig 2: Clinical signs in CME cases showing (a) Emaciation in an young rottweiler breed; (b) epistaxis; (c) Blanched mucosa; (d) Mucopurulent nasal discharge; (e) ecchymoses in the chest and axilla and (f) ecchymoses on the ventral abdomen of a young great dane breed.


       
Radiography (Fig 3), USG (Fig 4) and CT scan (Fig 5) of abdomen revealed hepatic, splenic and pulmonary changes. Splenic congestion was observed as hypoechoic area than liver, myelolipoma as hyper echoic mass with homogenous echo texture in spleen, distended renal pelvis and mineralization seen (hyper echogenic), kidney with indistinct cortico-medullary junction and small intestinal mucosa thickening (hyper echogenic). The CT scan in one chronic case was suggestive of lymphoma of the liver with an increased attenuation scale of 145 Hounsfield Units (HU), enlargement of spleen with an attenuation of 56 HU and inflammation of urinary bladder with an attenuation of 50 HU. Ultrasonographic study for the lymphoproliferative changes in liver, spleen and kidney in positive cases is reported to be more useful in supporting other diagnostic procedures especially during the subclinical phase (Singh et al., 2021a). Splenomegaly was observed in 25.0% of the CME cases (Table 1), whereas positivity was observed in 37.5% of the cases with splenomegaly (Table 2). These splenic changes were in accordance with Singh et al. (2021). Previously, Sarma et al. (2016) recorded splenomegaly in 8.33% of CME cases and in contrast, Angkanaporn et al. (2022) recorded in 76.9% of CME cases. Splenic enlargement could be associated with sequestration of ehrlichiae in infected macrophages (Waner and Harrus, 2013). Similar to this study, Cassaro et al. (2021) also reported splenic myelolipoma in an adult dog recovered from ehrlichiosis, though the neoplasm is reported to be rare in dogs. Renal, hepatic and pulmonary changes were observed in 41.6, 25.0 and 25.0 per cent, respectively of the CME cases (Table 1), whereas positivity was observed in 26.3, 21.4 and 25.0 per cent of the cases presented with renal, hepatic and pulmonary changes, respectively (Table 2). The hepato-renal changes observed in this study were in accordance with that of Singh et al. (2021a). Hepatic lymphoma in one of the positive cases could be associated with chronic inflammation and plasma cell proliferation caused by ehrlichiae resulting in development of B-cell lymphoma (Brunker and Hoove, 2007). These changes in liver, spleen and kidney could be due to immunological complications associated with immune dysregulation resulting in tissue injury (Singh et al., 2021a), since ehrlichia affects whole lymphatic system resulting in hyperplasia (Aziz et al., 2022). Inflammatory changes observed in other organs such as lungs, urinary bladder and intestinal mucosa might be due to the systemic inflammatory response (SIRS) caused by cytokines, acute phase protein and nitric oxide elicited by ehrlichiae (Asawapattanakul et al., 2021).

Fig 3: Radiography showing (a) Severely enlarged liver and spleen; (b) moderately enlarged spleen and (c) pulmonary infiltration in dogs affected with CME.



Fig 4: Ultrasonography showing (a) splenic congestion (hypoechoic area); (b) myelolipoma (hyper echoic area) of spleen; (c) mucosal thickening of small intestine (d) distended renal pelvis and mineralization (hyper echogenic area) and (e) indistinct cortico-medullary junction of kidney in CME positive cases.



Fig 5: Computerized tomography showing (a) lymphoma of the liver (scale of 145 HU); (b) enlargement of spleen (scale of 56 HU) and (c) inflammation of urinary bladder (a scale of 50 HU) in a CME positive case.



Table 2: Comparison of Haemato-biochemical values (Mean ± SE) of CME cases with control group.


       
Haematological values (Mean ± SE) in positive cases (Table 2) revealed a significant decrease (p<0.05) in haemoglobin (Hb) level and increase (p<0.05) in white blood cell (WBC) and monocyte count than control group. Serum biochemical values (Table 2) revealed an increase (p<0.05) in the blood urea nitrogen (BUN) level (p<0.01) and serum alkaline phosphatase (SAP) (p<0.05) than control group. Haematological changes (Table 1 and 2) such as anaemia, thrombocytopenia and neutropenia could be associated with the bone marrow hypoplasia caused by ehrlichiae in chronic form. Lymphocytosis, monocytosis and the resultant leucocytosis could be associated with the pathogen targeting mononuclear cells and lymphocytes (Asawapattanakul et al., 2021). However, lymphocytosis was reported to be the most common finding in the subclinical canine ehrlichiosis (Lorente et al., 2008). Serum biochemical changes (Table 2) such as increased levels of BUN, creatinine and phosphorus and decreased calcium level indicated chronic kidney disease (CKD). The CKD could be associated with immune mediated glomerulonephritis (IMGN) (Crivellenti et al., 2021) or SIRS specific to ehrlichiae resulting in increased C-reactive protein (CRP) and interleukin-6 (IL-6) levels (Asawapattanakul et al., 2021). Hypoalbuminemia might be due to vasculitis resulting in loss of proteins or hepatopathy (Singh et al., 2021; Gamit et al., 2024) and hyperglobulinemia as a sequel of strong humoral response associated with persistent ehrlichiae (Crivellenti et al., 2021).
       
The prevalence rate of ehrlichiosis with logistic regression analysis for various clinical variables was presented in Table 3. The prevalence rate was high in dogs with signs such as epistaxis, circling/ joint pain, emaciation, monocytosis, leucopenia and ecchymoses. The risk for the prevalence of CME was 5 to 6 times more in dogs with emaciation and melena, 3 to 4 times more with monocytosis, leucopenia, epistaxis and circling/ joint pain, 2 to 3 times with anaemia, thrombocytopenia and lymphadenopathy, than in dogs not associated with these clinical signs. A negative causal association was noticed for hepatic changes, haematochezia, vomiting, neutrophilia and no causal association was found in dogs with haemorrhagic diarrhoea, haematemesis and haematuria.

Table 3: Prevalence rate of ehrlichiosis with logistic regression analysis specific to various clinical signs (95% CI=28.2 to 76.22; df-15; P=0.0016) and host determinants (95% CI=7.07 to 66.07; df =21, P=0.015).


       
The specific prevalence rate (Table 3) was higher in dogs in the age group of 4-6 year (p>0.05) with two times the risk, females (p>0.05) with less than 2 times the risk, pure breeds (p<0.01) with 3 to 4 times the risk and recent exposure to ticks (p>0.05) with 2 to 3 times the risk, than in dogs of other groups. The increased risk in older dogs could be due to the increased exposure to the Ehrlichiae (Singh et al., 2021 and Aziz et al., 2022). In contrast, a high prevalence in dogs less than one year old (Kalaivanan et al., 2020), males (Asawapattanakul et al., 2021), both sex (Costa et al., 2007) and absence of sex preference (Asawapattanakul et al., 2021) was also reported. The high susceptibility in certain pure breeds could be associated with  increased preference to Rottweiler, Pitbull, Pug and Great Dane breeds by pet owners. In contrast, a high prevalence was recorded in German Shepherd (Kalaivanan et al., 2020 and Singh et al., 2021a), associated with their low cell mediated immune response (Dhankar et al., 2011) and in Labrador retriever (Pothiappan et al., 2024). More positivity in dogs with recent exposure to tick bites could be due to the seroconversion associated with an active ehrlichial infection.
               
In the forest tree analysis (Fig 6), the boxes represented the point estimates of RR and the whiskers represented the confidence interval for the variables. Since, the 95% CI from the pooled estimates (diamond) was entirely on the right side of no-effect line (corresponding to 1), the difference in the RR between the clinical variables was statistically significant. In contrast, 95% CI from the pooled estimates crossed the no-effect line indicating that there existed no significant difference. The graphical representation of prevalence of CME is shown in Fig 7.

Fig 6: Forest tree analysis of relative risk (CI: 95%) for (a) clinical findings and (b) host determinants drawn on a logarithmic scale in the prevalence of CME.



Fig 7: Graphical representation of prevalence of CME associated with the (a) spectrum of signs and (b) host determinants in dogs with GI bleeding signs.

CONCLUSION

Nearly one fourth of the dogs presented with gastro intestinal bleeding signs were found to be infected with E. canis, where majority of the cases were sub-acute and a few cases were chronic. Lateral flow assay as a point- of- care assay with on-spot diagnosis was useful in detecting the ehrlichia cases which were missed by conventional microscopy. Melena, monocytosis, lymphocytosis and leucocytosis were identified as early ehrlichia predictors in initiating the specific treatment to prevent the cases entering into the fatal chronic (myelosuppressive) form. Imaging techniques such as ultra sonography and computerized tomography could be useful in detecting the lymphoproliferative changes caused by ehrlichia in liver, spleen, kidney, lungs, urinary bladder and intestine.

ACKNOWLEDGEMENT

The authors would like to thank the Director of Clinics and Dean, Veterinary College and Research Institute, Namakkal, for facilitating this study.
 
Funding
 
The authors declare that no funds or grants were received during the preparation of this manuscript.

CONFLICT OF INTEREST

This study does not require approval of ethical committee. The authors have no relevant financial or non-financial interests to disclose.

REFERENCES


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    Full Research Article

    Prevalence of Ehrlichiosis in Dogs with Gastrointestinal Bleeding Signs and Meta-analysis of Causal Association

    S
    Saravanan Subramanian1,*
    K
    Kathirvel Shanmugam2
    S
    Sumathi Devarajan3
    D
    Dhivya Praba Ramasamy Abimanyu1
    S
    Selvaraju Ganapathy1
    1Department of Veterinary Public Health and Epidemiology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.
    2Department of Veterinary Surgery, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.
    3Department of Veterinary Clinical Medicine, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.

    ABSTRACT

    Background: Most of the canine monocytic ehrlichiosis (CME) cases are left undetected until they reach the chronic stage which is non-responsive to the treatment resulting in death associated with panleucopenia. Hence, diagnosis of clinical or subclinical CME is of paramount importance to prevent entering into the fatal chronic stage.

    Methods: Dogs (n=50) presented with haemorrhagic signs were subjected to microscopic examination and immunochromatography test. Positive cases were subjected to haemato-biochemical analysis, radiography, ultrasonography and computerized tomography. Meta-analysis of clinical variables was done to determine the causal association.

    Result: CME was observed in 22.0 per cent of cases by lateral flow assay (LFA) and 2.0 per cent by microscopic examination. Pathological changes were noticed in the liver, spleen, kidney, lungs, urinary bladder and intestine. A decrease (P<0.05) in haemoglobin level (8.66±1.59), increase (p<0.05) in white blood cell (21.87±5.2), monocyte count (8.5±1.06) and serum alkaline phosphatase level (151.33±48.12) and increase (p<0.01) in blood urea nitrogen (57.64±12.82) was observed. Melena (91.6%) was the predominant sign among positive cases. The sign- specific prevalence rate was high for epistaxis, circling/ joint pain (100.0%), emaciation (100.0%) and monocytosis (75.0%). The relative risk for the prevalence of CME was 5 to 6 times more in dogs with emaciation (95% CI, 2.26 to 12.18) and melena (95% CI, 0.73 to 6.71). High prevalence was observed in females (28.5%, p>0.05), 4-6-year-old (45.0%, p>0.05), pure breeds (100.0%, p<0.01) and recent tick infestation (31.2%, p>0.05). Antibody-based test, imaging techniques, haemato-biochemical analysis and the causal association could be useful tools in identifying the persistent ehrlichial infection.

    INTRODUCTION

    Canine monocytic ehrlichiosis (CME) is a significant vector borne disease caused by the obligate intracellular bacteria, Ehrlichia canis, transmitted by the tick, Rhipicephalus sanguineus and dogs act as reservoir (Aziz et al., 2022). The pathogen attacks macrophages and leucocytes in dogs (Mylonakis and Theodorou, 2017) and is reported to be zoonotic (Andric, 2014).
           
    The CME occurs as acute, subacute and chronic, with the clinical findings such as fever, lethargy, anaemia, lymphadenomegaly, hepatomegaly, petechiae or ecchymoses, vasculitis and extended bleeding period during estrus (Aziz et al., 2022). Some dogs due to ineffective treatment may become subclinical carriers for months or years by evasion of immune response (Greene at al., 2012). Most of dogs do not show any visible signs except mild thrombocytopenia (Maggi et al., 2014) and may enter into the most fatal chronic stage (myelosuppressive form). In chronic form, bleeding disorders such as ecchymoses, epistaxis, haematuria, melena and prolonged bleeding from venipuncture sites are more common (Mylonakis et al., 2019).
           
    The diagnosis of the ehrlichiosis becomes challenging owing to the several overlapping and non-specific clinical signs (Bai et al., 2017). Further, in recovered cases, the low ehrlichaemia is undetectable by molecular methods such as polymerase chain reaction (PCR) (Theodorou et al., 2013).  An early diagnosis would help in prompt treatment of cases of ehrlichiosis before entering into the fatal chronic stage (Aziz et al., 2022). Hence, the study is undertaken to report the early diagnosis of CME by lateral flow assay, to identify changes in the organs and to determine the causal association in the occurrence of CME.

    MATERIALS AND METHODS

    A cross-sectional study in the dogs (n=50) presented with the signs of gastrointestinal bleeding in 2023 to the Veterinary Clinical Complex, Veterinary College and Research Institute, Namakkal was conducted. Serum samples were subjected to lateral flow immuno assay based immunochromatography test (Pet X®, J and B Biotech Ltd, UK) for detection of E. canis antibodies. The positive result was indicated by the red lines both in control (C) and test (T) zones, whereas, negative result was indicated by the red line only in C zone. Giemsa stained peripheral blood smears were subjected to microscopic examination.
           
    The prevalence rate was statistically analysed between groups by Chi-square test. Blood were collected for haemato-biochemical analysis by auto Veterinary hematology Analyzer (Rayto®, Version 2.4e, RT7600Vet, Rayto Life and Analytical Sciences Co., Ltd., China) and auto Biochemistry analyzer (A15 Biosystems SA, Spain and the values were compared with healthy control group by Student t-test (using SPSS software). Radiography, ultrasonography (USG) (Esoate®, Italy) and computerized tomography (CT) (Toshiba Alexion 16 Slice CT scanner, Japan) were performed in positive cases. The causal association for the clinical variables was determined by logistic regression analysis using Med calc Statistical software and a forest tree analysis of RR (CI:95%) was plotted on a logarithmic scale.

    RESULTS AND DISCUSSION

    Microscopic examination (× 100) could detect morulae of ehrlichia within monocytes (Fig 1) in one case (2.0 per cent), whereas sandwich lateral flow assay could detect positivity (Fig 1) in 11 cases (22.0 per cent) and an overall prevalence of 24.0 per cent was observed for CME in dogs. Kappa analysis revealed only a slight agreement (Kappa value=0.18) between the tests (Landis and Koch, 1977). Ehrlichia could not be detected in most of the cases owing to the poor sensitivity of microscopic examination (Singh et al., 2021; Angkanaporn et al., 2022), as detection of morulae could be possible only in 4 to 6% of cases (Aziz et al., 2022; Sarawade et al., 2023; Gamit et al., 2024 and Checa et al., 2024). Previously, Senthil et al. (2023) recorded a highest prevalence of 56.37 per cent of E. canis in dogs from Chennai city. In this study, the immunochromatography test could detect more ehrlichia infections before entering into chronic form. Serological tests could detect both current and past ehrlichia infections and molecular test is recommended to distinguish the active infection (Sainz et al., 2015). Immuno chromato- graphy test (ICT) is reported to be a quick and useful diagnostic tool with 87% sensitivity and 95% specificity (Geromichalou and Faixova, 2017). Previously, Checa et al. (2024) also reported a high prevalence of CME by LFA.

    Fig 1: (a) Microscopic examination of Giemsa stained blood smears showing morulae of E. canis within monocyte (´ 100); (b)Lateral flow assay based immunochromatography test showing positivity for CME and (c) LFA showing negativity for CME.


           
    The clinical manifestations of positive cases are presented in Table 1 and Fig 2. From Table 1, most of the cases (84.4%) were found to be sub-acute as they showed clinical signs without fever (Waner and Harrus, 2013). Melena, anaemia, lymphadenopathy and thrombocytopenia were the most common signs observed in the positive cases which concurred to the findings of Singh et al. (2021). The bleeding signs could be associated with anti-platelet antibodies or bone marrow suppression or vasculitis (Asawapattanakul et al., 2021; Aziz et al., 2022). A very few were chronic cases (16.6%) showing panleucopenia, as chronic CME is indicated by severe pancytopenia due to myelosuppression caused by the ehrlichiae (Sainz et al., 2015; Singh et al. 2021). 

    Table 1: Distribution of signs in the positive cases of CME with gastrointestinal bleeding signs.



    Fig 2: Clinical signs in CME cases showing (a) Emaciation in an young rottweiler breed; (b) epistaxis; (c) Blanched mucosa; (d) Mucopurulent nasal discharge; (e) ecchymoses in the chest and axilla and (f) ecchymoses on the ventral abdomen of a young great dane breed.


           
    Radiography (Fig 3), USG (Fig 4) and CT scan (Fig 5) of abdomen revealed hepatic, splenic and pulmonary changes. Splenic congestion was observed as hypoechoic area than liver, myelolipoma as hyper echoic mass with homogenous echo texture in spleen, distended renal pelvis and mineralization seen (hyper echogenic), kidney with indistinct cortico-medullary junction and small intestinal mucosa thickening (hyper echogenic). The CT scan in one chronic case was suggestive of lymphoma of the liver with an increased attenuation scale of 145 Hounsfield Units (HU), enlargement of spleen with an attenuation of 56 HU and inflammation of urinary bladder with an attenuation of 50 HU. Ultrasonographic study for the lymphoproliferative changes in liver, spleen and kidney in positive cases is reported to be more useful in supporting other diagnostic procedures especially during the subclinical phase (Singh et al., 2021a). Splenomegaly was observed in 25.0% of the CME cases (Table 1), whereas positivity was observed in 37.5% of the cases with splenomegaly (Table 2). These splenic changes were in accordance with Singh et al. (2021). Previously, Sarma et al. (2016) recorded splenomegaly in 8.33% of CME cases and in contrast, Angkanaporn et al. (2022) recorded in 76.9% of CME cases. Splenic enlargement could be associated with sequestration of ehrlichiae in infected macrophages (Waner and Harrus, 2013). Similar to this study, Cassaro et al. (2021) also reported splenic myelolipoma in an adult dog recovered from ehrlichiosis, though the neoplasm is reported to be rare in dogs. Renal, hepatic and pulmonary changes were observed in 41.6, 25.0 and 25.0 per cent, respectively of the CME cases (Table 1), whereas positivity was observed in 26.3, 21.4 and 25.0 per cent of the cases presented with renal, hepatic and pulmonary changes, respectively (Table 2). The hepato-renal changes observed in this study were in accordance with that of Singh et al. (2021a). Hepatic lymphoma in one of the positive cases could be associated with chronic inflammation and plasma cell proliferation caused by ehrlichiae resulting in development of B-cell lymphoma (Brunker and Hoove, 2007). These changes in liver, spleen and kidney could be due to immunological complications associated with immune dysregulation resulting in tissue injury (Singh et al., 2021a), since ehrlichia affects whole lymphatic system resulting in hyperplasia (Aziz et al., 2022). Inflammatory changes observed in other organs such as lungs, urinary bladder and intestinal mucosa might be due to the systemic inflammatory response (SIRS) caused by cytokines, acute phase protein and nitric oxide elicited by ehrlichiae (Asawapattanakul et al., 2021).

    Fig 3: Radiography showing (a) Severely enlarged liver and spleen; (b) moderately enlarged spleen and (c) pulmonary infiltration in dogs affected with CME.



    Fig 4: Ultrasonography showing (a) splenic congestion (hypoechoic area); (b) myelolipoma (hyper echoic area) of spleen; (c) mucosal thickening of small intestine (d) distended renal pelvis and mineralization (hyper echogenic area) and (e) indistinct cortico-medullary junction of kidney in CME positive cases.



    Fig 5: Computerized tomography showing (a) lymphoma of the liver (scale of 145 HU); (b) enlargement of spleen (scale of 56 HU) and (c) inflammation of urinary bladder (a scale of 50 HU) in a CME positive case.



    Table 2: Comparison of Haemato-biochemical values (Mean ± SE) of CME cases with control group.


           
    Haematological values (Mean ± SE) in positive cases (Table 2) revealed a significant decrease (p<0.05) in haemoglobin (Hb) level and increase (p<0.05) in white blood cell (WBC) and monocyte count than control group. Serum biochemical values (Table 2) revealed an increase (p<0.05) in the blood urea nitrogen (BUN) level (p<0.01) and serum alkaline phosphatase (SAP) (p<0.05) than control group. Haematological changes (Table 1 and 2) such as anaemia, thrombocytopenia and neutropenia could be associated with the bone marrow hypoplasia caused by ehrlichiae in chronic form. Lymphocytosis, monocytosis and the resultant leucocytosis could be associated with the pathogen targeting mononuclear cells and lymphocytes (Asawapattanakul et al., 2021). However, lymphocytosis was reported to be the most common finding in the subclinical canine ehrlichiosis (Lorente et al., 2008). Serum biochemical changes (Table 2) such as increased levels of BUN, creatinine and phosphorus and decreased calcium level indicated chronic kidney disease (CKD). The CKD could be associated with immune mediated glomerulonephritis (IMGN) (Crivellenti et al., 2021) or SIRS specific to ehrlichiae resulting in increased C-reactive protein (CRP) and interleukin-6 (IL-6) levels (Asawapattanakul et al., 2021). Hypoalbuminemia might be due to vasculitis resulting in loss of proteins or hepatopathy (Singh et al., 2021; Gamit et al., 2024) and hyperglobulinemia as a sequel of strong humoral response associated with persistent ehrlichiae (Crivellenti et al., 2021).
           
    The prevalence rate of ehrlichiosis with logistic regression analysis for various clinical variables was presented in Table 3. The prevalence rate was high in dogs with signs such as epistaxis, circling/ joint pain, emaciation, monocytosis, leucopenia and ecchymoses. The risk for the prevalence of CME was 5 to 6 times more in dogs with emaciation and melena, 3 to 4 times more with monocytosis, leucopenia, epistaxis and circling/ joint pain, 2 to 3 times with anaemia, thrombocytopenia and lymphadenopathy, than in dogs not associated with these clinical signs. A negative causal association was noticed for hepatic changes, haematochezia, vomiting, neutrophilia and no causal association was found in dogs with haemorrhagic diarrhoea, haematemesis and haematuria.

    Table 3: Prevalence rate of ehrlichiosis with logistic regression analysis specific to various clinical signs (95% CI=28.2 to 76.22; df-15; P=0.0016) and host determinants (95% CI=7.07 to 66.07; df =21, P=0.015).


           
    The specific prevalence rate (Table 3) was higher in dogs in the age group of 4-6 year (p>0.05) with two times the risk, females (p>0.05) with less than 2 times the risk, pure breeds (p<0.01) with 3 to 4 times the risk and recent exposure to ticks (p>0.05) with 2 to 3 times the risk, than in dogs of other groups. The increased risk in older dogs could be due to the increased exposure to the Ehrlichiae (Singh et al., 2021 and Aziz et al., 2022). In contrast, a high prevalence in dogs less than one year old (Kalaivanan et al., 2020), males (Asawapattanakul et al., 2021), both sex (Costa et al., 2007) and absence of sex preference (Asawapattanakul et al., 2021) was also reported. The high susceptibility in certain pure breeds could be associated with  increased preference to Rottweiler, Pitbull, Pug and Great Dane breeds by pet owners. In contrast, a high prevalence was recorded in German Shepherd (Kalaivanan et al., 2020 and Singh et al., 2021a), associated with their low cell mediated immune response (Dhankar et al., 2011) and in Labrador retriever (Pothiappan et al., 2024). More positivity in dogs with recent exposure to tick bites could be due to the seroconversion associated with an active ehrlichial infection.
                   
    In the forest tree analysis (Fig 6), the boxes represented the point estimates of RR and the whiskers represented the confidence interval for the variables. Since, the 95% CI from the pooled estimates (diamond) was entirely on the right side of no-effect line (corresponding to 1), the difference in the RR between the clinical variables was statistically significant. In contrast, 95% CI from the pooled estimates crossed the no-effect line indicating that there existed no significant difference. The graphical representation of prevalence of CME is shown in Fig 7.

    Fig 6: Forest tree analysis of relative risk (CI: 95%) for (a) clinical findings and (b) host determinants drawn on a logarithmic scale in the prevalence of CME.



    Fig 7: Graphical representation of prevalence of CME associated with the (a) spectrum of signs and (b) host determinants in dogs with GI bleeding signs.

    CONCLUSION

    Nearly one fourth of the dogs presented with gastro intestinal bleeding signs were found to be infected with E. canis, where majority of the cases were sub-acute and a few cases were chronic. Lateral flow assay as a point- of- care assay with on-spot diagnosis was useful in detecting the ehrlichia cases which were missed by conventional microscopy. Melena, monocytosis, lymphocytosis and leucocytosis were identified as early ehrlichia predictors in initiating the specific treatment to prevent the cases entering into the fatal chronic (myelosuppressive) form. Imaging techniques such as ultra sonography and computerized tomography could be useful in detecting the lymphoproliferative changes caused by ehrlichia in liver, spleen, kidney, lungs, urinary bladder and intestine.

    ACKNOWLEDGEMENT

    The authors would like to thank the Director of Clinics and Dean, Veterinary College and Research Institute, Namakkal, for facilitating this study.
     
    Funding
     
    The authors declare that no funds or grants were received during the preparation of this manuscript.

    CONFLICT OF INTEREST

    This study does not require approval of ethical committee. The authors have no relevant financial or non-financial interests to disclose.

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