Pathology in Practice

Steven J. Kruzeniski Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.

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Fiona M. Tam Department of Small Animal Clinical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.

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Hilary J. Burgess Department of Veterinary Pathology, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.

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History

A 16-week-old sexually intact male Basenji-cross dog, imported from the upper west region of Ghana, was evaluated at the Veterinary Medical Center at the Western College of Veterinary Medicine as part of a wellness examination. At 4 weeks of age, while still in Ghana, the puppy had a body condition score of 1 on a scale from 1 to 5 (1 = emaciated; 5 = obese), was 6% dehydrated, was parasitized by numerous ticks (species not identified), and had multiple circular areas of alopecia. At that time, the puppy was treated with 100 mL of lactated Ringer's solution SC, anthelmintic medication (oral administration; type unknown), antifungals (topical administration; type unknown), and ivermectin (0.2 mg/kg [0.09 mg/lb], SC, twice 14 days apart). Two months later, with the exception of failure to grow, the puppy was in better health and body condition and was imported directly to Saskatchewan, Canada. During the interval between arriving in Saskatchewan at 12 weeks of age and the evaluation at 16 weeks of age, the puppy's progress was monitored by the owner, with no further improvement noted.

Clinical and Clinicopathologic Findings

On physical examination, the puppy was bright, alert, and responsive but was noticeably small for its age at 4.12 kg (9.1 lb). It had a body condition score of 2.5 (scale from 1 to 5), pale mucous membranes, and enlarged submandibular, prescapular, and inguinal lymph nodes. Blood (anticoagulated with EDTA) and serum samples were submitted to Prairie Diagnostic Services, Saskatoon, SK, Canada, for a CBC and serum biochemical analysis. The erythrogram revealed moderate normocytic, hypochromic, regenerative anemia (Hct, 0.22 L/L [reference interval, 0.365 to 0.573 L/L]; hemoglobin concentration, 72 g/L [reference interval, 128 to 196 g/L]; RBC count, 3.32 × 1012 RBCs/L [reference interval, 5.2 × 1012 RBCs/L to 8.2 × 1012 RBCs/L]; and absolute reticulocyte count, 239 × 109 reticulocytes/L [reference interval, < 80 × 109 reticulocytes/L]). There was also mild leukocytosis (20.4 × 109 leukocytes/L; reference interval, 4.8 × 109 leukocytes/L to 13.9 × 109 leukocytes/L) characterized by a mild left shift (0.20 × 109 band neutrophils/L; reference interval, ≤ 0.1 × 109 band neutrophils/L), mild toxic change in the neutrophil cell line, moderate lymphocytosis (10.6 × 109 lymphocytes/L; reference interval, 1.2 × 109 lymphocytes/L to 5.0 × 109 lymphocytes/L), and mild monocytosis (1.22 × 109 monocytes/L; reference interval, 0.08 × 109 monocytes/L to 1.0 × 109 monocytes/L). Platelets could not be accurately enumerated because of clumping.

On examination of a blood smear routinely stained with a modified Wright stain, 3 neutrophils were noted to contain large, pale staining, ellipsoid structures with a poorly staining nucleus (Figure 1). Five hundred neutrophils were counted in a buffy coat preparation, indicating a 0.6% prevalence of these inclusions. Examination of the buffy coat preparation revealed rare mononuclear cells containing variably sized, round, granular basophilic cytoplasmic inclusions.

Figure 1—
Figure 1—

Photomicrographs of a peripheral blood direct smear (A) and buffy coat preparation (B) from a 16-week-old Basenji-cross dog that had been imported from the upper west region of Ghana and was evaluated as part of a wellness examination. In panel A, notice a neutrophil that contains a large, pale staining, ellipsoid structure with a poorly staining nucleus. Modified Wright stain; bar = 20 μm. In panel B, the central lymphocyte contains a round, granular basophilic cytoplasmic inclusion. Modified Wright stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 243, 12; 10.2460/javma.243.12.1705

The most important findings of the serum biochemical analysis included mildly high activities of alkaline phosphatase (129 U/L; reference interval, 9 to 90 U/L), alanine aminotransferase (63 U/L; reference interval, 19 to 59 U/L), glutamate dehydrogenase (14 U/L; reference interval, 0 to 7 U/L), and sorbitol dehydrogenase (8.0 U/L; reference interval, 0 to 4 U/L); moderate hypoalbuminemia (21 g/L; reference interval, 28 to 38 g/L); and mild hyperglobulinemia (39 g/L; reference interval, 23 to 37 g/L).

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →

Additional Clinicopathologic Findings

Direct smears from fine-needle aspirate samples of the enlarged lymph nodes were microscopically evaluated. The samples contained a heterogeneous lymphoid population, of which most were small, well-differentiated lymphocytes with few neutrophils, plasma cells, and macrophages admixed. Etiologic agents were not discernible in the examined smears. The cytologic appearance was consistent with mild lymphoid hyperplasia with resultant clinical lymphadenopathy. The anticoagulated blood sample was assessed for Dirofilaria immitis antigen and antibodies against Ehrlichia canis, Borrelia burgdorferi, and Anaplasma phagocytophilum with a point-of-care ELISA.a A positive result for anti–E canis antibodies correlated with the observation of morulae within the mononuclear population on buffy coat examination. Although there is variable cross-reaction between E canis and Ehrlichia chaffeensis with the point-of-care ELISA,1 a PCR assay was not performed to differentiate the species; however, infection of dogs with E chaffeensis has only convincingly been identified in the United States,2 thereby excluding it as a potential differential diagnosis. Results for D immitis antigen and antibodies against B burgdorferi and A phagocytophilum were negative.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis and case summary: mixed Hepatozoon canis and E canis infection in a dog.

Comments

Hepatozoon canis and Ehrlichia canis are tick-borne pathogens that share a common vector, specifically Rhipicephalus sanguineus.2–5 The tick species that parasitized the dog of the present report in Ghana were not identified; however, R sanguineus is known to be one of the many tick species in that country.6 It has been documented that individual ticks can carry > 1 type of infectious agent,7 but the distinctly different life cycles of H canis and E canis and the dog's high level of tick exposure suggested that > 1 tick was likely responsible for this mixed infection.

Ehrlichia canis multiplies within hemocytes and salivary epithelium and eventually infects the midgut epithelium of its tick host4; subsequently, it is transmitted during a blood meal. In contrast, after ingestion by a tick, H canis (an apicomplexan) undergoes fertilization and sporogony within the digestive system, followed by release of oocysts into the hemocoel.3,8 The oocysts contain a membrane that envelops numerous sporocysts in which the infective sporozoites are found.3,8 Sporozoites do not migrate to the feeding parts or salivary gland of the tick and therefore cannot be transmitted through subsequent blood meals.3,5 Rather, infected ticks are themselves ingested, with the digestion process rupturing oocysts and releasing sporozoites into a dog's intestinal tract.9 Sporozoites migrate to the liver, spleen, lymph nodes, and bone marrow for merogony, although the route of dissemination is not clear.3 It is at this stage where the lifecycle of H canis differs from that of Hepatozoon americanum, which is geographically limited, at this time, to the United States. In contrast to H canis, H americanum merogony takes place within skeletal or cardiac muscle fibers.10,11 Although a PCR assay was not performed to differentiate these 2 species of Hepatozoon in the case described in the present report, evidence of merogony in tissues was not pursued, and gamonts of the 2 Hepatozoon spp detected in peripheral blood samples cannot be visually distinguished, the dog's geographic origin limited the diagnosis to H canis.12 In addition, the clinical signs and additional laboratory findings were also more typical of H canis infection.

Whereas H canis often causes subclinical to mild disease, with only a few heavily infected dogs developing a more severe form, H americanum infection typically has a severe and debilitating disease course, reflecting its tissue tropism, where the patient develops fever, mucopurulent ocular discharge, generalized bone and muscle pain, and lameness.13–16 Laboratory findings for the dog described in the present report correlated well with those typically associated with H canis infection, although the high alkaline phosphatase activity could also be attributed to the bone isoform in a young growing dog, rather than a result of H canis infection alone. High activities of hepatocellular leakage enzymes (alanine aminotransferase, glutamate dehydrogenase, and sorbitol dehydrogenase) are not commonly evident in dogs with H canis infection; however, merogony and subsequent release of merozoites can result in hepatitis.9 Observation of morphologically characteristic gamonts during microscopic evaluation of stained blood smears is the most common method for detecting H canis infection8 and was the method of diagnosis in the present case. The case described in the present report reinforces the importance of blood smear evaluation as part of a patient's minimum database, considering that the clinical signs and laboratory data would not have otherwise specifically implicated H canis infection in this dog.

A grading scheme based on the number of clinical signs (lethargy, anorexia, weight loss, oculonasal discharge, lymphadenopathy, skin and coat alterations, and pale mucous membranes) has previously been used for dogs infected with H canis.14 Specifically, those patients with 1 or 2 clinical signs are designated as having a mild form of disease, those with 3 or 4 clinical signs are designated as having a moderate form, and those with 5 to 7 clinical signs are designated as having a severe form.14 In that study,14 the degree of parasitemia generally correlated well with the grading scheme. On the basis of that grading scheme, the dog of the present report would have been classified as having a moderate form of the disease; however, the degree of parasitemia (< 1%) was more consistent with a mild form (degree of parasitemia, 0% to 3%). Coinfections have been commonly identified in H canis–infected dogs with moderate and severe disease, and it has been suggested that clinical signs may be more appropriately attributed to the concurrent pathogens or potentially an interaction among pathogens.14,17 Therefore, evaluation for presence of additional infections should be considered in H canis–infected dogs with clinical signs.14 In the case described in the present report, coinfection with E canis may have accounted for the discrepancy between the grading scheme classification and degree of H canis parasitemia.

The dog of the present report was treated with doxycycline (5 mg/kg [2.3 mg/lb], PO, q 12 h) for 30 days, during which time 2 doses of imidocarb dipropionate (5 mg/kg, SC, q 14 d) were also administered. A CBC was repeated at the end of the treatment period, with resolution of all abnormalities. At that time, neither H canis gamonts nor E canis morulae were discernible in stained blood smears. The dog's energy level increased, and the growth rate improved; eventually, the dog's weight and stature were considered normal given its breed and age. One year after treatment, the dog remained well without recurrence of clinical or hematologic abnormalities.

a.

Canine SNAP 4Dx, Idexx Laboratories Inc, Westbrook, Me.

References

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