Cytological evaluation of bone marrow in 11 cattle with enzootic bovine leukosis

Abstract

As the frequency and significance of bone marrow (BM) infiltration in enzootic bovine leukosis (EBL) remain unclear, this study evaluated BM involvement and its relationship with peripheral blood (PB) findings in 11 cattle with EBL using PB evaluation, and BM aspiration cytology. BM infiltration was identified in 5 of 11 (45%) EBL cases based on BM smears. All EBL cases showed circulating atypical lymphocytes, although BM infiltration was not consistently present. Two cases with extreme PB lymphocytosis showed BM infiltration. Cytopenia was variably observed and was most severe in cases with extensive BM involvement. This study provides combined assessment of PB and BM changes in EBL cattle and suggests that such evaluation enables a more comprehensive characterization of hematological alterations.

Enzootic bovine leukosis (EBL) is a transmissible neoplastic disease caused by bovine leukemia virus (BLV), which is widely distributed among cattle herds and remains endemic in many countries. In Japan, the seroprevalence of BLV infection has been reported to be 40.9% in dairy cattle and 28.7% in beef breeding cattle [12]. BLV primarily infects B lymphocytes, and approximately 30% of BLV-infected cattle develop persistent lymphocytosis (PL). Of those animals, only a small proportion (1–5%) eventually develop malignant B-cell lymphoma, referred to as EBL, which typically involves multiple organs and has a poor prognosis [2]. The clinical manifestation of EBL is nonspecific, and cytomorphological evaluation and molecular analysis are often required for diagnosis. In small animals and humans, bone marrow (BM) evaluation plays a crucial role in staging multicentric lymphoma and diagnosing leukemia [1], and the presence of atypical lymphocytes in peripheral blood (PB) may clinically suggest lymphoma infiltration into the BM [3, 11, 14, 15]. In contrast, BM aspiration is not routinely performed in cattle with lymphoma, and its diagnostic or clinical significance in relation to PB findings remains unclear. Although several studies have described the distribution of neoplastic lesions in bovine leukosis [9, 10], including BM involvement, the frequency and clinical significance of BM infiltration in BLV-associated lymphoma have not been fully elucidated [13, 14, 17]. Therefore, this study aimed to evaluate the clinical utility of BM examination in cattle with EBL by comparing BM and PB findings.

Cattle submitted to the Obihiro University of Agriculture and Veterinary Medicine (OUAVM) for necropsy between March 2017 and September 2018 were included in this study. Animals were classified into EBL and non-EBL groups based on the following inclusion criteria; positive for BLV by nested PCR and a histopathological diagnosis of lymphoma for the EBL group, and no hematological abnormalities based on complete blood count (CBC) and a histopathological diagnosis of non-neoplastic, non-inflammatory disease for the non-EBL group. CBCs were performed using EDTA blood samples collected from the jugular vein on the day of arrival at OUAVM using an automated hematology analyzer (Sysmex, Kobe, Japan). PB smear evaluation was performed using the same samples stained with Wright–Giemsa stain (Muto Pure Chemicals, Tokyo, Japan). Genomic DNA was extracted from whole blood or FNA samples from superficial lymph nodes or any affected lesions. Nested PCR for detecting BLV was conducted, targeting the 5′ long terminal repeat (LTR) region of the BLV genome according to the method described by a previous report [8]. BM aspiration was conducted just before necropsy under general sedation, which was induced with a combination of xylazine (0.2 mg/kg IV) and thiamylal (5 mg/kg IV) [8]. The BM aspiration procedure followed a standard protocol [6]. A Jamshidi BM biopsy needle (8 G × 10 cm, Fuji Systems, Tokyo, Japan) was used to aspirate BM fluid from the sternum. BM smears were stained with Wright–Giemsa stain. All animals were euthanized under anesthesia for necropsy after BM aspiration, and histopathological examinations were conducted by anatomical pathologists at the Department of Pathology, OUAVM. Each animal was euthanized in accordance with ethical and animal welfare requirements under the Guidelines for the Care and Use of Agricultural Animals of Obihiro University (Approval 18-38).

Hematology results were categorized as follows according to a textbook: anemia (hematocrit (Hct) <22%), lymphocytosis (lymphocyte count >7.5 × 10⁹/L), neutropenia (neutrophil count <1.8 × 10⁹ /L), and thrombocytopenia (platelet count <193 × 10⁹/L) [6]. A differential count of 100 PB leukocytes was performed in all cases, and the absolute leukocyte count was calculated based on the total white cell count obtained by automated hematology analyzer. The PB infiltration of neoplastic lymphocytes was defined as the presence of atypical lymphocytes detected during the differential count of leukocytes that exhibited atypical features, including intermediate to large cell size (>2 times of RBC), a high nucleus-to-cytoplasm ratio (N/C), and nuclear atypia such as pleomorphism, irregular nuclear shape, and prominent or multiple nucleoli [6]. BM evaluation involved assessing cellularity, degree of megakaryopoiesis under low power magnification, followed by a marrow leukocyte differential count under high power magnification. A total of 500 nucleated cells were classified into 8 myeloid categories (myeloblast, promyelocyte, myelocyte, metamyelocyte, band neutrophil, segmented neutrophil, eosinophil, and basophil), 5 erythroid categories (rubriblast, prorubricyte, basophilic rubricyte, polychromatophilic rubricyte, and metarubricyte), as well as lymphocytes and monocytes. BM infiltration of neoplastic lymphocytes was defined as the presence of lymphocytes >6% of all nucleated cells (ANC) accompanied by cytomorphological atypia [6]. The atypical features were defined using the same cytological criteria as those of PB.

A total of 16 cattle were included in this study. Eleven cattle were classified into the EBL group (1 male and 10 female), and 5 cattle were classified into the non-EBL group (all female). Median age was 3.9 years old (range: 1.6–11.2 years old) in the EBL group and 2.5 years old (range: 1.1–6.6 years old) in the non-EBL group. Four cattle in the EBL group were Japanese Black, and the remaining 7 and all non-EBL cattle were Holstein (Supplementary Table 1). On hematology, lymphocytosis was noted in 6 cases, neutropenia in 4 cases, anemia in 4 cases, and thrombocytopenia in 1 case (Supplementary Table 2). Two cases showed bi-cytopenia (anemia with neutropenia) and 1 case showed pancytopenia. PB infiltration of atypical lymphocytes was observed in all 11 cases in the EBL group. The atypical lymphocytes noted in all cases were mostly intermediate- to large-sized and had scant to small amounts of pale cytoplasm, with round, irregular, or convoluted nuclei with finely stippled chromatin and occasional prominent nucleoli (Supplementary Fig. 1).

The percentage of lymphocytes in BM varied considerably among cases (Table 1). A total of 8 cases had lymphocyte proportions greater than 6% of all nucleated cells (Nos. 3–11). Of these, 5 cases (Nos. 7–11) also had atypical lymphocytes and were therefore considered to have BM infiltration. The atypical lymphocytes were intermediate to large cells with high N/C ratio, round to irregular nuclei shape, finely stippled to coarse chromatin, and occasional prominent nucleoli (Fig. 1). Among the 6 cases with lymphocytosis on CBC, BM infiltration was identified in only 3 cases. In contrast, 2 cases with BM infiltration showed no evidence of lymphocytosis. Notably, BM infiltration was present in both cases with extreme PB lymphocytosis (>150 × 10⁹/L) (Fig. 2). Although all cases in the EBL group had PB infiltration, obvious BM infiltration was confirmed in 5 of the 11 cases. Of the 5 cases with BM infiltration, 3 showed peripheral blood cytopenia, including 2 cases with bi-cytopenia (anemia and neutropenia) and 1 case with pancytopenia. The two cases with the highest proportions of BM infiltration (49.6% and 90.2%) exhibited bi-cytopenia and pancytopenia, respectively.

Table 1. Marrow leukocyte differential percentages of 11 enzootic bovine leukosis (EBL) and 5 non-EBL cases.

EBL Case No.	1	2	3	4	5	6	7	8	9	10	11	Non-EBL*
Myeloblast (%)	2.2	7.4	9.8	1.4	0.6	1.4	4.8	0.4	1.2	0.8	0.0	0.2	–	5.6
Promyelocyte (%)	6.0	12.4	5.2	2.0	0.8	2.6	4.4	2.4	1.6	0.8	0.2	1.6	–	5.0
Myelocyte (%)	12.0	5.4	8.2	2.8	1.6	2.2	3.6	5.0	3.8	2.6	0.6	4.0	–	20.8
Metamyelocyte (%)	14.0	18.2	17.4	1.4	6.6	7.8	9.6	8.0	3.2	7.4	0.4	6.0	–	19.6
Neutrophil band (%)	24.0	13.2	11.4	10.2	9.8	11.2	7.0	10.8	10.2	12.4	1.4	10.2	–	24.0
Neutrophil band (%)	14.8	1.4	2.0	16.8	4.4	19.2	4.0	2.4	6.6	2.0	1.4	1.4	–	19.0
Eosinophil (%)	2.0	4.4	5.2	1.8	13.0	3.8	3.6	0.0	9.4	1.8	0.4	5.8	–	10.6
Basophil (%)	0.2	0.0	0.0	0.0	0.6	0.4	0.2	0.0	3.0	0.0	0.0	0.0	–	0.4
Rubriblast (%)	0.0	0.0	0.0	0.0	0.1	0.1	0.0	0.2	0.0	0.0	0.0	0.0	–	0.2
Prorubricyte (%)	1.6	3.8	2.8	5.0	5.7	5.7	3.2	6.4	7.0	3.6	0.8	3.4	–	6.4
basophil rubricyte (%)	2.0	1.2	0.8	0.0	1.8	0.0	3.2	1.4	0.0	0.0	0.2	0.0	–	2.6
Polychromatrophilic rubricyte (%)	11.8	10.8	10.0	26.4	30.4	11.8	17.4	24.2	26.4	9.4	2.0	12.6	–	30.8
Metarubricyte (%)	5.0	16.8	21.6	23.8	16.0	21.4	13.0	22.8	13.8	8.8	2.4	8.8	–	14.6
Lymphocyte (%)	3.6	3.6	5.0	7.8	8.4	9.8	15.0	15.2	13.4	49.6	90.2	0.0	–	1.6
Monocute (%)	0.8	1.4	0.8	0.0	0.8	2.6	3.4	0.8	0.4	0.8	0.0	0.0	–	0.8
M/E	3.6	1.2	1.7	0.7	0.7	1.3	1.2	0.5	0.8	1.3	0.8	1.0	–	2.7
Atypical lymphocyte	-	-	-	-	-	-	+	+	+	+	+

*: Range (lowest−highest) in 5 non-EBL cases. -: cattle without bone marrow infiltration, +: cattle with bone marrow infiltration.

Fig. 1.

Bone marrow smear from enzootic bovine leukosis case No. 7. Wright–Giemsa stain. Increased numbers of atypical lymphocytes (white arrows).

Fig. 2.

Relationship between peripheral blood lymphocyte count, bone marrow lymphocyte percentage, and bone marrow infiltration (Black dots; cattle with bone marrow infiltration, white dots; cattle without bone marrow infiltration, dashed line; the upper reference limit of the normal lymphocyte count [6].

In the present study, BM infiltration was detected in approximately 45% of cases with EBL. It was previously reported that 53% of cases with lymphosarcoma showed increased lymphocyte proportions in BM, however, those lymphocytes lacked cytological atypia, and the findings were attributed to PB contamination [17]. In the present study, BM infiltration was defined not only based on increased lymphocyte proportions but also the presence of cytological atypia and the blood contamination was minimal, allowing distinction between more accurate BM infiltration. In cases Nos. 3–6, the lymphocytes were predominantly small and lacked significant cytological atypia. Therefore, these cases were not interpreted as BM infiltration, despite lymphocyte proportions exceeding 6%. Although comparable metastatic BM infiltration has been described in other species, such as FeLV-infected cats with T-cell lymphoma, to the best of our knowledge, this is the first study documenting BM infiltration of lymphoma in a case series of cattle with EBL [5]. Our findings also indicate that PB lymphocytosis and circulating atypical lymphocytes are not consistently associated with BM infiltration in cattle with EBL. Only half of the cases with lymphocytosis showed BM infiltration, and only 45% of cases with PB infiltration showed BM infiltration. These results suggest that mechanisms other than BM infiltration, such as the accumulation of long-lived lymphocytes with transcriptionally silenced viruses or circulating neoplastic lymphocytes from lesions other than BM, may play a role in the development of lymphocytosis in EBL [4]. In contrast, two cases with extreme PB lymphocytosis (>150 × 10⁹/L; Case Nos. 7 and 11) showed marked BM infiltration, implying that extensive marrow involvement may contribute severe leukocytosis in a subset of animals. Cytopenia was observed in 3 of 5 cases with BM infiltration, and 1 of 2 bi-cytopenic (anemia with neutropenia) cases had no evidence of BM infiltration, supporting that BM infiltration is not always associated with peripheral cytopenia. However, 2 cases with the greatest degree of BM infiltration (Nos. 10 and 11) showed bi-cytopenia or pancytopenia, likely reflecting myelophthisis caused by replacement of normal hematopoietic tissue by neoplastic cells. Although pancytopenia has been reported in horses and dogs with acute lymphocytic leukemia, to our knowledge, this is the first report documenting pancytopenia associated with BM infiltration in cattle with EBL [7, 16]. Overall, the inconsistency between PB findings and BM findings indicates that BM examination serves to provide additional information on disease extent in cases with EBL, particularly in cases with inconclusive or atypical PB abnormalities.

While this study provides new insights into BM findings in cattle with EBL, several limitations should be acknowledged. Most importantly, BM evaluation was based solely on aspiration cytology without histopathological or molecular confirmation of neoplastic infiltration in BM, including clonality assessment, which may have led to underestimation of BM infiltration due to the limitations of morphological assessment alone. Future studies incorporating BM histopathology together with immunophenotypic, molecular, and clonality analyses, as well as flow cytometry, in larger case series provide a more comprehensive and objective assessment of BM and PB involvement in cattle with EBL.

Conflicts of Interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.