An Unusual Presentation of Granulocytic Anaplasmosis in a Young Dog

Introduction

Canine granulocytic anaplasmosis, caused by infection with bacterium of the family Anaplasmataceae, is diagnosed in many areas of the United States, particularly in dogs residing in the upper midwestern, northeastern, and western states. This disease is also found in several European countries.¹ This case report describes a 20 wk old puppy that presented with physical examination and laboratory findings atypical of canine granulocytic anaplasmosis. Doxycycline therapy was prescribed and all hematologic and physical abnormalities resolved.

Case Report

A 20 wk old, 6.4 kg, intact male, mixed-breed puppy was presented to Ocean State Veterinary Specialists for evaluation of a 3 day history of progressive lethargy and inappetence. The patient was also showing signs consistent with nausea (i.e., pica, hypersalivation) and had one episode of vomiting 3 days prior to presentation. Pale mucous membranes were noted by the owner prior to presentation. The patient had been purchased approximately 1 mo earlier from a breeder in Rhode Island. Insect bites and a tick of unknown species had been found on the patient by the referring veterinarian. The puppy had been administered parvovirus and Borrelia burgdorferi vaccinations the day prior to the onset of his clinical signs. He had not been in contact with any other domestic animals. A monthly topical flea and tick preventative was also given.

A complete blood count (CBC), serum biochemistry panel, and 4DX SNAP^a test were performed by the referring veterinarian prior to referral. Abnormalities on the automated CBC included a low red blood cell count (3.35×10⁶/μL; reference range, 4.7–8.5×10⁶/μL), low hematocrit (20.5%; reference range, 32.0–55.0%), low hemoglobin concentration (6.6 g/dL; reference range, 10.3–18.0 g/dL), low total WBC count (4.27×10³/μL; reference range, 5.5–16.9×10³/μL), low absolute neutrophil count (0.83×10³/μL; reference range, 3.0–12.0×10³/μL), and low platelet count (23.0×10³/μL; reference range, 175–500×10³/μL). Abnormalities on the serum biochemistry profile included hypoalbuminemia (2.1 g/dL; reference range, 2.5–4.4 g/dL), hypoproteinemia (5.3 g/dL; reference range, 5.4–8.2 g/dL), hyperphosphatemia (7.5 mg/dL; reference range, 2.9–5.6 mg/dL), and hyperglycemia (121 mg/dL; reference range, 60–110 mg/dL). A positive result for Anaplasma phagocytophilum was obtained on the 4DX SNAP test.

A review of the CBC and blood smear by a medical technologist certified by the American Society for Clinical Pathology confirmed the anemia, thrombocytopenia, and neutropenia. Large platelets were seen, potentially indicative of active thrombopoiesis. There was a mild increase in blasts, most consistent with lymphoblasts, but blasts of a myeloid origin could not be ruled out at that time. Blast nuclei were atypical in appearance and multiple, distinct nucleoli were seen.

On initial examination at the Ocean State Veterinary Specialists, the rectal temperature was 39.1°C, heart rate was 144 beats/min, and respiratory rate was 54 breaths/min. The puppy was in good body condition and had an adequate muscle mass for his age. Mucous membranes were pale and slightly tacky, indicating approximately 5% dehydration. Capillary refill time was <2 sec. No petechiae or ecchymoses were noted. Mentation was generally dull, but no neurologic deficits were present.

A repeat CBC at the time of presentation confirmed a persistent pancytopenia with worsening of the hematocrit (16%) and platelet count (16.0×10³/μL). A blood smear with a manual differential count confirmed the presence of neutropenia, thrombocytopenia, and nonregenerative anemia. No morphologic abnormalities were noted in the erythrocytes. The packed cell volume was 20% and total solids, determined by refractometer, were 4.0 g/dL. The serum was clear. A parvovirus SNAP test^b was negative. An abdominal ultrasound was performed, which revealed a mild amount of free peritoneal fluid. Thoracic radiographs were within normal limits.

An ultrasound-guided aspirate of the abdominal fluid was obtained and submitted for cytology. The fluid was moderately opaque with an orange color. The specific gravity by refractometer was 1.018 and the total solids were 2.0 g/dL. The WBC count was 13.0×10³/μL and the red blood cell count was 20.0×10³/μL. A moderate to heavy mixture of nondegenerate neutrophils and foamy macrophages was seen. There were rare mature lymphocytes. Prominent morulae were present within the cytoplasm of many of the neutrophils (Figure 1). Due to the prior hematologic observation of circulating blasts, a bone marrow aspirate and core biopsy was performed from the right iliac crest. The bone marrow smears were of normal cellularity and contained intact marrow particles and dispersed hematopoietic cells. Cytology of the marrow aspirate showed a mild increase in megakaryocytes. Active myelopoiesis (39%) and increased erythropoiesis (48%) were noted. Full maturation was seen in both cell lines. The myeloid to erythroid ratio was approximately 0.8:1.0. There were 12% lymphoid cells and 1% plasma cells. The majority of the lymphoid cells were normal, small lymphocytes, with a rare atypical cell observed. These cells were most likely reactive in nature. The bone marrow core biopsy tissue was moderately cellular with hyperplasia of the erythroid, myeloid, and lymphoid cell lines present.

Due to the possibility of a lymphoproliferative disorder based on the pathology review of the peripheral blood and bone marrow aspirate, polymerase chain reaction for antigen receptor rearrangement assay was performed on both a peripheral blood sample and a sample of bone marrow. Both samples tested negative.

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A serum titer for A. phagocytophilum was consistent with active infection (1:640; reference range, <1:20). The puppy was started on a course of oral doxycycline (5 mg/kg q 12 hr) to treat the anaplasmosis. Treatment with enrofloxacin (5 mg/kg per os q 24 hr) was also initiated to provide gram negative coverage in the face of neutropenia. IV fluids (lactated Ringer's solution with 20 mEq/L potassium chloride) were given to correct for the assessed dehydration and to provide maintenance fluid support.

A CBC was performed on each day of hospitalization. The total WBC count had normalized after the first day (6.08×10³/μL), but the absolute neutrophil count had decreased to 0.44×10³/μL by the second day. By the third day, the neutrophil count increased to 0.88×10³/μL. Both the hematocrit and the packed cell volume increased during the patient's stay in the hospital. By the third day, the hematocrit was 22.6%, the packed cell volume was 26%, and total solids (via refractometer) were 5.6 g/dL. The absolute reticulocyte count was 74.1×10³/μL (reference range, <80.0×10³/μL). The platelet count gradually increased, and 3 days after admission it was 70.0×10³/μL.

As a result of the hematologic and clinical improvement, the patient was weaned from the IV fluids and discharged 4 days after admission. Doxycycline and enrofloxacin were continued.

A CBC, performed a few days after discharge, showed progressive improvement in hematologic data. A CBC performed 8 days after discharge from the hospital showed that the neutrophil count was within normal limits. The enrofloxacin therapy was discontinued at that time. By 15 days after discharge, all hematologic values were within normal limits. According to the owners, the puppy was completely normal.

Discussion

A. phagocytophilum, a bacterium of the family Anaplasmataceae, is a gram negative organism that is an obligate aerobe and intracellular parasite. Infection with A. phagocytophilum in dogs is commonly referred to as canine granulocytic anaplasmosis. A. phagocytophilum is transmitted in the United States by the ticks Ixodes scapularis and I. pacificus. The bacteria enter neutrophils by endocytosis and are then incorporated into the phagosomes. The bacteria then replicate by binary fission, forming the microscopically visible morulae.² Rarely, morulae have been noted in eosinophils.³

Various case studies, as well as retrospective and prospective studies, have reported and evaluated the numerous clinicopathologic abnormalities associated with dogs affected by canine granulocytic anaplasmosis. To the authors’ knowledge, finding peritoneal effusion is not a previously noted sequela of canine granulocytic anaplasmosis. The lack of morulae in the peripheral blood with the presence of prominent morulae in the peritoneal fluid is also unusual.

The age of this puppy and the changes in white blood cell lines are not typical of the disease. Infection with A. phagocytophilum has been noted in juvenile patients; however, it is relatively uncommon. In a study of dogs affected by canine granulocytic anaplasmosis in Minnesota and Wisconsin, only 3% were <1 yr.⁴ A study was conducted at the University of Berlin where 258 sick dogs were evaluated. Of these dogs, 18 were infected with A. phagocytophilum with no concurrent infections. The age of the dogs ranged from 2 yr to 13 yr.² One study of 34 dogs in Minnesota did, however, show that 24% of the dogs evaluated were <1 yr, with the youngest dog 1 mo.⁵ The patient in the case presented herein was 5 mo at the time of presentation.

Tick infestation had been noted by the owner. The incubation period for A. phagocytophilum varies from 1 wk to 2 wk. A recent case report failed to demonstrate perinatal transmission of A. phagocytophium from an infected bitch to her offspring.⁶ As such, typical tick vectored transmission was likely, as opposed to either perinatal transmission that has been described in humans or transplacentary infection documented in cattle.^7,8 The patient in this case had not received prior transfusions, so transmission via infected blood was considered highly unlikely.

Peritoneal effusion has not been previously reported to be a sequela of infection with A. Phagocytophilum in dogs. There have been reports of A. phagocytophilum infection in horses that have caused abdominal effusion with morulae seen within the neutrophils of the effusion. Severe vasculitis with petechial, ecchymotic, and intracavitary hemorrhage has also been seen in horses infected with A. phagocytophilum.^1,9 Vasculitis is a potential explanation for the abdominal effusion seen in this case, induced by immune-antigen complex deposition and/or the production of proinflammatory cytokines by neutrophils infected by A. phagocytophilum.¹⁰ The remainder of the abdominal ultrasound was unremarkable, with no hepatomegaly, splenomegaly, or other definitive signs of inflammation seen. Also, no signs typically associated with vasculitis, such as ventral (dependent) edema, erythema, petechiations, or echymoses were seen on physical examination. The albumin was initially below the reference interval, but it had not reached a level generally considered to be low enough for spontaneous effusion secondary to decreased oncotic pressure to occur.¹¹ No signs of portal hypertension, hepatic congestion, or other causes for increased hydrostatic pressure were identified.

The lack of morulae in the peripheral blood combined with the presence of prominent neutrophilic morulae in the peritoneal fluid is also atypical. It is possible that the peritoneal effusion developed subsequent to the initial hematologic acute infection. Morulae may have been more readily visualized in the phagosomes of peripheral neutrophils if blood samples had been analyzed earlier in the disease process.

Various permutations of anemia, thrombocytopenia, and leukopenia have been reported previously.² Thrombocytopenia has been seen in >80% of cases.^4,12,13 Proposed mechanisms include increased platelet consumption secondary to disseminated intravascular coagulation, splenic sequestration, immune-mediated platelet destruction, or production of inhibitory factors by A. phagocytophilum.^2,14–16 Less than 50% of dogs have a mild to moderate, nonregenerative, normochromic, normocytic anemia, suggestive of anemia of inflammation.^2,17 Other potential pathologic mechanisms for anemia in infected dogs include immune-mediated erythrocyte destruction and inhibition of hematopoiesis secondary to A. phagocytophilum cytokine production.¹

Leukopenia, characterized by lymphopenia and eosinopenia, is also a common finding; however, neutrophil counts are rarely outside of the reference intervals.^4,12,13 Leukopenia was a hematologic abnormality seen in this case, but it was characterized by a neutropenia. Both the lymphocyte and the eosinophil counts were within normal limits throughout treatment. Given that neutrophils are specifically targeted by A. phagocytophilum for replication and dissemination, neutrophil consumption via lysis is a potential explanation for the neutropenia; however, bone marrow suppression should also be considered as a possible cause.

Due to the initial clinical pathologic findings of pancytopenia and blast cells seen in the peripheral circulation, hematologic neoplasias such as acute leukemia or hematogenous lymphoma were included on the initial list of differential diagnoses. Given the relative rarity of hematogenous neoplasms in juvenile patients combined with the negative polymerase chain reaction for antigen receptor rearrangement assay, the hyperplastic bone marrow aspirates with no obvious neoplastic cells, and a response to doxycycline therapy, a diagnosis of cancer was highly unlikely. The abnormal lymphoid and myeloid cells seen in circulation were most likely reactive cells, lymphoblasts, or other precursors prematurely released from the bone marrow secondary to bone marrow disease and/or active regeneration.

Conclusion

Canine granulocytic anaplasmosis is a disease that can potentially affect several body systems and can account for a wide variety of clinicopathologic abnormalities. In light of the clinical (i.e., lethargy, nausea, vomiting, and pale mucous membranes) and laboratory (i.e., anemia, thrombocytopenia, leukopenia, neutrophilic morulae, and high A. phagocytophilum titer) findings, this case was consistent with active A. phagocytophilum infection. As such, inclusion of canine granulocytic anaplasmosis on lists of differential diagnoses is warranted in cases displaying cavitary effusions.