A canine case of Ehrlichia canis infection without a history of being in an endemic area in Japan

Abstract

A mixed-breed, 8-year-old male dog developed neutropenia, thrombocytopenia, and hyperglobulinemia. Bone marrow hyperplasia and splenic plasmacytosis were cytologically observed. The dog had never been outside of Tokyo or Shizuoka Prefecture. Splenectomy was performed to confirm and remove the cause of splenic plasmacytosis. A histopathological diagnosis of splenic plasmacytoma was made; however, serum protein electrophoresis showed polyclonal gammopathy. Further screening was performed, and Ehrlichia canis infection was confirmed. The dog was treated with doxycycline for 5 weeks. After the antibiotic therapy, no relapse of neutropenia, thrombocytopenia, hyperglobulinemia, or positive polymerase chain reaction result of E. canis infection was observed for 3 years. Careful attention should be given to ehrlichiosis when exploring the cause of pancytopenia or hyperglobulinemia, regardless of the travel history.

Canine ehrlichiosis, including Ehrlichia canis infection, is a tick-borne disease with a worldwide distribution. E. canis infection has been reported to induce pancytopenia and/or fever, and chronic infection of E. canis also results in hyperglobulinemia, anemia, and/or thrombocytopenia [10]. E. canis infection has only been reported in dogs brought in from endemic countries into Japan [1, 11]. Therefore, Japan is considered a non-endemic area, and E. canis infection is not usually included in the differential diagnosis of dogs with no history of being in endemic areas [1]. Here, we report a canine case with E. canis infection, which had never been away from Tokyo or Shizuoka Prefecture.

An 8-year-old mixed Toy Poodle and Maltese male dog was referred to the Veterinary Teaching Hospital of Azabu University (VTH-AU) in July 2020. The dog was adopted by the present owner from a domestic breeder and has lived in Tokyo. It was occasionally brought to Shizuoka Prefecture; however, it had never been out of Tokyo or Shizuoka Prefecture. Although asymptomatic thrombocytopenia and hyperproteinemia had been detected persistently for 3 years (Table 1), it has not received any treatment. In addition, the dog had a history of infection with tick or mite 3 months prior to initial presentation to VTH-AU.

Table 1. Hematological parameters of the present case.

Day	WBC (/µL)	Seg (/µL)	RBC (×104/µL)	PCV (%)	PLT (×104/µL)	TP (g/dL)	ALB (g/dL)	PCR*	Antibody**	Treatment
−1,469	11,200	NE	738	53.2	27.6	7.2	NE	NE	NE
−1,127	6,000	NE	772	53.9	16.3	7.4	NE	NE	NE
−771	4,400	NE	698	47.8	6.5	8.4	NE	NE	NE
−385	3,600	NE	687	46.2	11.5	9.8	NE	NE	NE
−24	4,100	NE	621	39.9	4.7	11.2	NE	NE	NE
0	4,080	1,810	622	40.4	8.3	11.3	2.4	NE	NE
14	3,730	1,980	559	36.1	8.2	10.9	2.5	NE	+***	Splenectomy
15	10,360	8,860	493	32.9	14.1	9.9	2.4	NE	NE
30	9,010	5,100	543	35.1	27.5	7.5	2.7	+	NE
45	8,730	3,480	534	35.6	36.0	8.4	2.7	NE	NE	Doxycycline started
73	8,350	2,810	575	37.8	69.4	7.6	2.8	−	NE
80	NE	NE	NE	NE	NE	NE	NE	NE	NE	Doxycycline discontinued
175	7,330	2,500	631	41.6	59.5	7.2	3.0	−	NE
486	8,400	2,870	625	42.4	49.7	7.2	3.4	−	+
1,179	9,010	5,280	619	41.9	49.6	6.8	3.7	−	+

ALB: albumin, NE: not evaluated, PCR: polymerase chain reaction, PCV: packed cell volume, PLT: platelet, RBC: red blood cell, Seg: segmented neutrophil, TP: total protein, WBC: white blood cell, +: positive, –: negative. *RealPCR test; IDEXX Laboratories, Inc., Westbrook, ME, USA. **SNAP 4Dx Plus Test; IDEXX Laboratories, Inc. ***Evaluated at day 30 using the stored serum sample.

Routine blood tests were performed by using XT-2000iV (Sysmex Corp., Kobe, Japan) and Cobas^® 6000 (Roche Diagnostics K.K., Reinach, Switzerland), and revealed neutropenia (1,810/µL), thrombocytopenia (83,000/µL), and hyperglobulinemia (TP 11.3 g/dL, Alb 2.4 g/dL) (Table 1). Proteinuria was not detected (urine protein/creatinine ratio: 0.13). Radiography and ultrasonography revealed a splenomegaly with diffuse heterogeneous hypoechogenicity; however, no other abnormality was detected. Serum protein electrophoresis indicated polyclonal gammopathy. Liver and splenic fine needle biopsy and bone marrow aspiration were performed on day 7 to detect the cause of neutropenia, thrombocytopenia, and hyperglobulinemia. Plasma cell proliferation was detected in the spleen, whereas the smear of bone marrow only showed generalized hyperplasia and hemophagocytosis (Fig. 1). No pathogens or neoplastic changes were observed from each sample. Therefore, splenectomy was performed on day 14 to confirm the cause of plasma cell proliferation and remove it directly. Diffusely proliferated plasma cells replacing the lymphoid follicle were detected histopathologically in the spleen (Fig. 2). They were positive for Ki-67; therefore, it was diagnosed as plasmacytoma. A multicolor GeneScan analysis [4] on the splenic sample detected a clonal rearrangement of the T cell receptor γ-chain (TCR-γ) gene.

Fig. 1.

Bone marrow smear. Wright−Giemsa stain. (A) Apparent bone marrow hyperplasia was observed. Scale bar=100 µm. (B) Hemophagocytosis was observed (arrowhead), whereas plasma cell proliferation was not observed. Scale bar=10 µm.

Fig. 2.

Spleen histopathology. Hematoxylin and eosin stain. (A) The lymphoid follicle was replaced with the diffusely proliferated plasma cell. Scale bar=100 µm. (B) The plasma cells had little mitotic activity or atypia. Scale bar=20 µm.

On day 30, the neutropenia, thrombocytopenia, and hyperglobulinemia were resolved. However, the discrepancy between the histopathological diagnosis “plasmacytoma” and the findings of polyclonal gammopathy or clonal rearrangement of the TCR-γ gene remained. Therefore, screening polymerase chain reaction (PCR) analysis of several pathogens (RealPCR test; IDEXX Laboratories, Inc., Westbrook, ME, USA) from peripheral blood was performed, which revealed an E. canis infection. In addition, the dog was also seropositive against E. canis (SNAP 4Dx Plus Test; IDEXX Laboratories, Inc.). Therefore, the dog was further diagnosed with canine monocytic ehrlichiosis.

Based on the final diagnosis, doxycycline (11 mg/kg, q24 hr) was prescribed on day 45. On day 73, the PCR analysis of E. canis from peripheral blood was negative, and the doxycycline was discontinued on day 80. Later, the dog did not receive any treatment; however, it did not show any relapse of the E. canis infection, neutropenia, thrombocytopenia, or hyperglobulinemia for over 3 years.

Further PCR analysis was performed by using the stored samples of bone marrow, spleen, liver, and splenic lymph node with the primers including EHR16SD and EHR16SR under the previously reported protocol [9], and 16S rRNA gene of E. canis was detected in all samples. To perform a phylogenetic analysis, 16S rRNA gene was amplified using fD1/Rp2 primers [3, 7] and subjected to commercial laboratory (Eurofines Genomics, Bayern, Germany) for direct sequence. Phylogenic analysis was performed by using CLC Sequence Viewer 8 (QIAGEN, Hilden, Germany), and the detected gene sequence from the present case was close to the strain reported from Israel (Genbank accession number: U26740) (Fig. 3).

Fig. 3.

Phylogenic analysis constructed by the neighbor-joining method based on sequences of the 16S rRNA gene and was supported by 1,000 bootstrap replications.

This is the first report of a canine case without a history of being in an endemic area in Japan. Canine ehrlichiosis with E. canis infection reportedly develops with acute and/or chronic phases [8]. Acute disease usually develops within 2−4 weeks following tick transmission. Many E. canis-infected dogs undergo a subclinical infection phase and remain chronically infected for months or even years [8]. The present case was adopted from a breeder in Japan and had no history of being in endemic areas; therefore, it was infected with E. canis within Japan. Unfortunately, information of the present case’s parents was not available, therefore, the infection route (ixodid-mediated or vertical transmission) in this case remains unclear. However, the dog started to develop thrombocytopenia 3 years prior, and a tick or mite infection was documented after developing thrombocytopenia and hyperglobulinemia. Therefore, the pathogen is speculated to have already been spread around Tokyo.

Bone marrow hypoplasia has been documented to occur in canine monocytic ehrlichiosis in chronic severe form, one of the major mechanisms of pancytopenia [5, 6, 10]. However, this case showed bone marrow hyperplasia without any clinical symptoms; therefore, it was considered a reactive change against neutropenia and/or thrombocytopenia and would be under the subclinical phase of canine monocytic ehrlichiosis. Therefore, other reported mechanisms, including sequestration, increased consumption, and secretion of platelet-migration inhibition factor from lymphocytes [8], would result in the observed neutropenia and thrombocytopenia.

The present case was diagnosed as splenic plasmacytoma histopathologically; however, the association of splenic plasmacytoma and E. canis infection remains unknown. Extensive plasma cell infiltration into parenchymal organs in the acute phase of canine monocytic ehrlichiosis has been reported [5, 13]. A clonal rearrangement of TCR-γ gene was observed in the splenic sample of the present case. Similar findings had been reported in E. canis-infected dogs without malignancy, which was considered to indicate the presence of an antigens-specific T-cell population [2, 12]. Therefore, chronic E. canis infection might affect the antigen-specific reactions of T-cells as well as plasma cells, and further investigations on the association between them are warranted.

Interestingly, the neutropenia, thrombocytopenia, and hyperglobulinemia improved just after the splenectomy. This is consistent with a previous study performing splenectomy on dogs with experimental E. canis infection [6]. In this report, eight healthy dogs were inoculated with E. canis, of which four were splenectomized 4 weeks prior. The hematological parameters were recorded for 60 days. During the monitoring period, the pancytopenia and fever were milder in the splenectomized group than in the control group. From the findings, the spleen was essential in the pathogenesis of canine monocytic ehrlichiosis [6]. Therefore, splenectomy could be an effective option in treating canine monocytic ehrlichiosis, which warrants further investigation.

Since the present case had never been to endemic areas, we did not rule out ehrlichiosis at initial diagnosis. However, as described above, careful attention should be given to the disease when exploring the cause of pancytopenia or hyperglobulinemia, regardless of travel history.

CONFLICT OF INTEREST

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the study.