Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog

Mathios E. Mylonakis, Alex F. Koutinas, Gad Baneth, Zoe Polizopoulou, Anna Fytianou
Abstract: A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunouorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the rst reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The ndings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs. (Vet Clin Pathol. 2004;33:249251)
2004 American Society for Veterinary Clinical Pathology

Key Words: Anaplasmosis, cytology, ehrlichiosis, hepatozoonosis, rickettsia

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Mixed infections with multiple tick-borne pathogens are seen occasionally in dogs1,2 and often pose a diagnostic dilemma.3 No single diagnostic method has been accepted as the sole means of conrming tick-borne diseases. Therefore, clinical, clinicopathologic, serologic, and molecular examinations should be pursued in such cases for a denitive diagnosis.1 In this report, the clinicopathologic ndings in a dog simultaneously infected with Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum are described, emphasizing the value of buffy coat, bone marrow, and lymph node cytology in the diagnosis of mixed tickborne infections. A 5-month-old, female, fully vaccinated mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with a history of depression and anorexia for the past 48 hours. At physical examination, fever (40.38C), depression, peripheral lymphadenopathy, splenomegaly, and bilateral seromucous ocular and nasal discharges were detected. No ticks were found, although they had been occasionally witnessed in the past. A CBC (QBC Vet Autoread, IDEXX, Westbrook, ME, USA) was performed on EDTA-anticoagulated blood, and

Giemsa-stained smears were evaluated for cell morphology and a 100-cell differential WBC count. Hematology ndings revealed mild nonregenerative anemia (PCV 27.6%, corrected reticulocyte percentage 1.9%) and mild thrombocytopenia (115,000 platelets/lL, megathrombocytes 7%) (Table 1). Megathrombocyte percentage was determined by counting platelets 5 lm in diameter, as measured with an eyepiece micrometer, during evaluation of 200 platelets in Giemsa-stained blood smears. No serum biochemical or urinalysis abnormalities were found. Microscopic examination of 1000 elds per tissue using a 3100 oil immersion objective on Giemsa-stained buffy coat, bone marrow, and lymph node aspirate specimens revealed Ehrlichia sp morulae in monocytes, lymphocytes, and neutrophils and H canis gamonts in neutrophils (Figures 1 and 2). Buffy coat smears appeared of higher diagnostic yield, with a total of 2 Ehrlichia sp morulae in mononuclear cells, 5 morulae in neutrophils, and 100 H canis gamonts in neutrophils per 1000 elds, compared with bone marrow (3 morulae in monocytes) and lymph node (2 morulae in lymphocytes and 4 H canis gamonts in neutrophils) smears.4 In 2 neutrophils, both morulae and gamonts were observed

From the Clinic of Companion Animal Medicine (Mylonakis, Koutinas) and the Laboratory of Clinical Diagnosis and Clinical Pathology (Polizopoulou, Fytianou), School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece; and the School of Veterinary Medicine, Hebrew University of Jerusalem, Jerusalem, Israel (Baneth). This case was presented in the Case Review Session at the 5th Annual Meeting of the ESVCP-ECVCP, Uppsala, Sweden, September 3, 2003. Corresponding author: Dr A. F. Koutinas, Clinic of Companion Animal Medicine, School of Veterinary Medicine, Aristotle University of Thessaloniki, PO Box 16039, 54401, Thessaloniki, Greece (sanimed@vet.auth.gr). 2004 American Society for Veterinary Clinical Pathology

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Mixed Tick-Borne Infection in a Dog

Table 1. Hematology results in a dog with monocytic (Ehrlichia canis) and granulocytic (presumptive Anaplasma phagocytophilum) ehrlichiosis, and hepatozoonosis (Hepatozoon canis).
Parameter PCV (%) Hemoglobin (g/dL) MCHC (g/dL) Corrected reticulocyte (%) WBC (/lL) Platelets (/lL) Segmented neutrophils (/lL) Band neutrophils (/lL) Lymphocytes (/lL) Eosinophils (/lL) Monocytes (/lL) Basophils (/lL) *indicates not determined. Day 1 27.6 9.6 34.8 1.9 7400 115,000 4292 148 1406 444 1110 0 Day 28 35.3 12.4 35.2 * 7900 250,000 5100 120 2000 300 380 0 Reference Interval 3755 1218 30.036.9 <2 600017,000 200,000500,000 300011,000 0300 10004800 1001250 1501350 0100

Figure 2. Ehrlichia canis morula in a mononuclear leukocyte. Buffy coat smear, Giemsa, 3100 objective.

together in the same cell (Figure 1). Bone marrow particles were normocellular. An average of 4 megakaryocytes was found per 310 objective eld. The dog was seropositive for E canis (IgG reciprocal titer 1:200, cut-off value 1:100) but was negative for A phagocytophilum (E equi antigen) using immunouorescence assays (IFA).4,5 The dog also tested seropositive to H canis IgG by an ELISA technique (optical density 0.850, cut-off value for negative result  0.55).6 A nested polymerase chain reaction (PCR) was performed on EDTA-treated bone marrow aspirate material stored frozen (208C) for 2 weeks until evaluated (Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine, Thessaloniki, Greece). The PCR resulted in amplication of E canis 16srRNA gene (389 base pair).4 This method was not applied for the detection of A phagocytophilum.

The combined cytologic, serologic, and PCR results conrmed a diagnosis of mixed E canis, H canis, and presumptive A phagocytophilum (formerly E equi) infection, the latter based on the observation of organisms in blood rather than on serologic results. The dog was treated with oral doxycycline (Ronaxan, Merial, Lyon, France) at a dosage of 5 mg/kg, twice daily, for 4 weeks. Imidocarb dipropionate (Imizol, Essex Animal Health, Friesoyth, Germany) also was administered subcutaneously at a dosage of 5 mg/kg, twice, 14 days apart. Follow up examinations documented a remarkable clinical recovery within the rst 48 hours witnessed by reappearance of appetite and defervescence, followed by resolution of the clinicopathologic abnormalities (Table 1) and the disappearance of parasitemia (based on screening 1000 3100 objective elds of buffy coat smears) at the end of the 4-week (day 28 from admission) treatment. Based on telephone contact with the dogs owner, the dog remained clinically healthy at least 6 months after treatment was completed.

Discussion
The clinical manifestations and clinicopathologic abnormalities described in this case are seen frequently in either monocytic or granulocytic canine ehrlichiosis7,8 as well as in hepatozoonosis caused by H canis infection.9 Therefore, in this patient, clinical disease might be attributed to any one of the 3 agents, singly or most probably in synergism with the coexistent agents. A denitive diagnosis of ehrlichial infection usually is based on serologic results and, more recently, on PCR.3 The difculty in interpreting serology results, especially in endemic areas such as Greece, and the limited availability of standardized PCR assays worldwide,10 may justify the use of cytology as an easily applied and inexpensive diagnostic alternative.4 Cytology has high sensitivity for the diagnosis of acute but not chronic

Figure 1. A presumptive Anaplasma phagocytophilum morula (arrow) and a Hepatozoon canis gamont in a neutrophil. Buffy coat smear, Giemsa, 3100 objective.

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Mylonakis, Koutinas, Baneth, Polizopoulou, Fytianou

infections with E canis,7 A phagocytophilum,5 and H canis.5,11,12 In a recent study, the diagnostic sensitivity of cytology based on the examination of 1000 3100 objective elds on buffy coat, lymph node, and bone marrow smears in 50 dogs with natural acute monocytic ehrlichiosis was found to be 66%, 60.9%, and 34%, respectively.4 The mild thrombocytopenia, increased number of megathrombocytes, normal number of megakaryocytes, and spectacular clinical and clinicopathologic response to specic treatments were suggestive of acute E canis infection,7 which may also explain the easily found E canis morulae in the 3 tissue smears. The presence of A phagocytophilum morulae in as many as 824% of circulating neutrophils has been reported in the acute phase of granulocytic ehrlichiosis.5,8 H canis gamonts may be observed in up to 90% of circulating neutrophils, but 15% of cells are infected in the majority of cases.9 The detection of 2 different types of inclusions in a single neutrophil, in this case of presumptive Anaplasma morulae and H canis gamonts, is extremely rare.1315 The species identity of the granulocytic morulae in this report was not pursued by molecular methods. However, despite the negative serology, they most likely were A phagocytophilum and not E ewingii, as the latter organism has not been documented outside the United States.3 The recent detection of A phagocytophilum in dogs in Greece by PCR12 and documentation of the presence of Ixodes ricinus, a vector of A phagocytophilum in the same country,16 further substantiate a diagnosis of anaplasmosis in the dog described here. The lack of a detectable antibody titer to A phagocytophilum in spite of the relevant positive cytology was not unexpected, as seroconversion may occur before, during, or after the appearance of neutrophilic inclusions.17 The antigenic diversity that probably occurs among organisms belonging to the same species in different geographic regions may be another reason for the failure to detect anti-A phagocytophilum antibodies by the IFA technique.7 To our knowledge, this is the rst reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The combined cytologic evaluation of buffy coat, lymph node, and bone marrow was helpful in establishing the initial diagnosis and facilitated institution of specic therapy before obtaining the results of further diagnostic tests, such as serology and PCR.
Acknowledgments
The authors are grateful to Dr Inger Lilliehook and the Swedish Veterinary Institute, Uppsala, Sweden, for the E equi (A phagocytophilum) serology.

References
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