EHRLICHIOSIS IN DOGS IMPORTANCE OF TESTING FOR CONTRIBUTING AUTHORS CASE 1: SWIGGLES INTRODUCTION WITH PERSISTENT LYMPHOCYTOSIS

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1 THE IMPORTANCE OF TESTING FOR EHRLICHIOSIS IN DOGS WITH PERSISTENT LYMPHOCYTOSIS Contributing Authors: Mary Anna Thrall, DVM, MS, DACVP Diana Scorpio, DVM, MS, DACLAM Ross University School of Veterinary Medicine Basseterre, St. Kitts, West Indies INTRODUCTION Testing for monocytic ehrlichiosis is warranted in dogs with unexplained persistent lymphocytosis, particularly in endemic areas. The list of causes of persistent lymphocytosis in dogs is quite short and includes leukemia, canine ehrlichiosis, and rarely, hypoadrenocorticism. Canine ehrlichiosis is endemic in St. Kitts, West Indies, and persistent lymphocytosis in affected dogs is quite common. The following case presentations are examples of dogs with canine ehrlichiosis and persistent lymphocytosis. CASE 1: SWIGGLES Swiggles, a male dog infested with ticks and assumed to be approximately three years of age, was presented to the Ross University School of Veterinary Medicine (RUSVM) community clinic for testing for ehrlichiosis, and was PCR positive. Complete blood counts were performed over a three-month period (Table 1). Abnormalities on 11/20 included leukocytosis due to lymphocytosis, and eosinophilia. The apparent thrombocytopenia on that day is erroneous due to platelet clumping, as can be observed on the platelet histogram (Figure 1). Large granular lymphocytes were observed (Figure 2). The manual differential count performed that day was also erroneous due to neutrophil agglutination likely due to hyperproteinemia and subsequent uneven distribution of leukocytes in the counting area of the blood film. Doxycycline therapy (10 mg/kg sid) was initiated on 1/26/15 and was continued for 28 days. A biochemical profile using the Abaxis Vet Scan VS2 was performed on 1/30/15. The only abnormalities noted were total protein of 9.6 g/dl (reference interval is ) and globulin concentration of 6.2 g/dl (reference interval is ). Biochemical profile was repeated on 2/27/15 revealing total protein of 8.2 g/dl and globulin concentration of 4.7g/dl, both within the reference interval. Table 1. Swiggles complete blood counts (Abnormalities are bolded) 11/20/14^ 1/30/15^ 2/13/15^ 2/27/15 Ref Interval PCV (%) MCV (fl) Total Protein (g/dl) TNCC* (x 103µl) Segmented neuts(x 103µl) Lymphocytes(x 103µl) Monocytes(x 103µl) Eosinophils(x 103µl) Platelets(x 103µl) 57.5** ^Technician observations re blood film exam: Rouleaux present (due to increased globulin); numerous large granular lymphocytes present; neutrophil agglutination present ** Platelet count is erroneously low due to platelet clumping (observed on blood film) 30 Vetcom Volume 56

2 Figure 1 Figure 2 Volume 56 Vetcom 31

3 CASE 2: SURPRISE Surprise, an intact female dog assumed to be approximately three years of age was presented to the RUSVM community clinic for testing for ehrlichiosis and was PCR positive. Complete blood counts were performed over a one-month period (Table 2). Abnormalities include lymphocytosis, increased total protein, and on 2/18/15, one day after she had aborted a dead puppy, increased band neutrophils (Table 2). Doxycycline therapy (10 mg/kg sid) was initiated on 1/30/15 and was continued for 28 days. A biochemical profile was performed on 1/30/15. The only abnormalities noted were total protein of 9.7 g/dl and globulin concentration of 7.2 g/dl. The biochemical profile was repeated on 2/27/15, and both the total protein and globulin continued to be above the reference interval (9.7 g/dl and 6.7 g/dl, respectively). Table 2. Surprise s complete blood counts 1/30/15^ 2/6/15^ 2/18/15 2/27/15^ Ref Interval PCV (%) MCV (fl) Total Protein (g/dl) TNCC* (x 103µl) Segmented neuts(x 103µl) Band Neutrophils (x 103µl) ** Lymphocytes(x 103µl) Monocytes(x 103µl) Eosinophils(x 103µl) Platelets(x 103µl) ^Technician observations re blood film exam: Rouleaux present (due to increased globulin); numerous large granular lymphocytes present ** Neutrophils and band neutrophils exhibited marked toxic change CASE 3: WHISKEY Whiskey, a male dog assumed to be approximately three years of age, was presented to the Ross University School of Veterinary Medicine (RUSVM) community clinic for testing for ehrlichiosis and was PCR positive. Complete blood counts were performed over a three-month period (Table 3). Abnormalities on 11/20/14 included lymphocytosis and eosinophilia. The apparent thrombocytopenia is erroneous due to platelet clumping. Large granular lymphocytes were observed. Doxycycline therapy (10 mg/kg sid) was initiated on 1/26/15 and continued for 28 days. A biochemical profile using the Abaxis Vet Scan VS2 was performed on 1/30/15. The only abnormalities noted were total protein of 11.3 g/ dl (reference interval, ) and globulin concentration of 8.7 g/dl (reference interval, ). Biochemical profile was repeated on 2/27/15 revealing a total protein of 10.5 g/ dl and globulin concentration of 7.7g/dl. 32 Vetcom Volume 56

4 Table 3. Whiskey s complete blood counts 11/20/14^ 1/23/15^ 2/13/15^ 2/27/15^ Ref Interval PCV (%) MCV (fl) Total Protein (g/dl) TNCC* (x 103µl) Segmented neuts(x 103µl) Lymphocytes(x 103µl) Monocytes(x 103µl) Eosinophils(x 103µl) Platelets(x 103µl) 18** 58** 98** ^Technician observations re blood film exam: Rouleaux present (due to increased globulin); many large granular lymphocytes present **Electronic platelet count erroneously low due to platelet clumping (observed on the blood film) DISCUSSION Canine monocytic ehrlichiosis, first reported in 1935, is caused by Ehrlichia canis, a small, coccoid, gram-negative bacterium transmitted by Rhipocephalus sanguineus, a ubiquitous tick. Clinical signs and lesions of canine ehrlichiosis are related to the infection and immune response produced by the host. It is known from small studies that dogs with ehrlichiosis have increased numbers of circulating large granular lymphocytes and a higher lymphocyte concentration than normal dogs (Lorente et al, 2008). Although chronic infectious disease of all types is often listed as a differential for lymphocytosis in dogs, a review of the literature suggests that with the exception of Ehrlichia canis infection, lymphocytosis is not a feature of canine chronic infectious disease (Avery et al, 2007). Numerous studies have shown that naturally occurring E. canis infection can result in lymphocytosis with values up to 17,000 lymphocytes/µl (Kuehn et al, 1985; Codner et al, 1986; Weiser et al, 1991; Frank et al, 1999; Heeb et al, 2003), although not all case series describe lymphocytosis (Breitschwerdt et al, 1998). Anecdotal reports and the experience of some clinicians and clinical pathologists suggest that lymphocyte counts up to 30,000 cells/µl are possible in E canis infection. The lymphocyte response usually consists of an increased percentage of cells with a large granular lymphocyte (LGL) phenotype (McDonough et al, 2000; Lorente et al, 2008), which were shown to be CD8+ T cells. Therefore, an important differential for lymphocytosis in dogs is E. canis infection, but the frequency with which E. canis is associated with lymphocytosis is not known. The lymphocytosis may persist for several months, even after treatment. Testing for ehrlichiosis is warranted in dogs with unexplained persistent lymphocytosis. However, in areas not endemic for ehrlichiosis, a neoplastic lymphoproliferative disorder is the most common cause of persistent lymphocytosis. Lymphoid leukemia may be seen with chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and lymphoma with circulating neoplastic cells (stage V lymphoma). Unlike ALL and stage V lymphoma, lymphocytes in dogs with CLL usually appear mature and normal. The diagnosis of CLL can sometime be made based on the magnitude of the lymphocytosis, since a lymphocyte concentration of greater than 35,000/µl would almost certainly be due to leukemia. In patients with a lymphocytosis of lesser magnitude, ehrlichiosis can be eliminated as a differential based on testing, and CLL can be confirmed using either immunophenotyping by flow cytometry or by clonality testing using the PCR for antigen rearrangement assay (PARR) (Avery et al, 2007). Volume 56 Vetcom 33

5 THE IMPORTANCE OF TESTING FOR EHRLICHIOSIS IN DOGS WITH PERSISTENT LYMPHOCYTOSIS These cases also emphasize the importance of blood film examination. For example, the thrombocytopenia reported on three of Whiskey s CBCs and the first CBC performed on Swiggles were erroneous due to platelet clumping, which was observed on the blood film. Another example is Surprise s CBC performed on 2/18/15. The presence of band neutrophils detected on the manual differential count were important in diagnosing significant inflammation, but they were not detected by the electronic cell counter, as band neutrophils cannot be differentiated from segmented neutrophils. On the other hand, the instrument may be reporting more reliable differential counts than can be obtained by a manual count if neutrophil agglutination is present, as was seen on Swiggles blood films on numerous occasions. References Avery AC, Avery PR. Determining the significance of persistent lymphocytosis. Vet Clin Small Anim. 2007;37: Breitschwerdt EB, Hegarty BC, Hancock SI. Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii. J Clin Microbiol 1998;36: Codner EC, Farris-Smith LL. Characterization of the subclinical phase of ehrlichiosis in dogs. J Am Vet Med Assoc 1986;189: Frank JR, Breitschwerdt EB. A retrospective study of ehrlichiosis in 62 dogs from North Carolina and Virginia. J Vet Intern Med 1999;13: Heeb HL, Wilkerson MJ, Chun R, et al. Large granular lymphocytosis, lymphocyte subset inversion, thrombocytopenia, dysproteinemia, and positive Ehrlichia serology in a dog. J Am Anim Hosp Assoc 2003;39: Kuehn NF, Gaunt SD. Clinical and hematologic findings in canine ehrlichiosis. J AmVet Med Assoc 1985;186: Lorente C, Sainz A, and Tesouro MA. Immunophenotype of Dogs with Subclinical Ehrlichiosis. Animal Biodiversity and Emerging Diseases: Ann. N.Y. Acad. Sci. 2008;1149: McDonough SP, Moore PF. Clinical, hematologic, and immunophenotypic characterization of canine large granular lymphocytosis. Vet Pathol 2000;37: Weiser MG, Thrall MA, Fulton R, et al. Granular lymphocytosis and hyperproteinemia in dogs with chronic ehrlichiosis. J Am Anim Hosp Assoc 1991;27: Vetcom Volume 56