A clinical case of enzootic bovine leukosis in a Holstein cow with minor clonality of B-cell in the peripheral blood

Abstract

A 4-year 9-month-old Holstein-Friesian dairy cow presented with anorexia. On physical examination, swelling of superficial lymph nodes, pelvic masses, and prolonged urination posture after urinating a small amount were noted. Hematological examination revealed no lymphocytosis. The bovine leukemia virus proviral load was relatively high. At necropsy, enlarged lymph nodes, a large mass in the pelvic cavity, and mass lesions in several organs were observed. Histopathological examination revealed the proliferation of neoplastic lymphocytes, which were immune-positive for CD79α and negative for CD3. B-cell clonality test indicated the presence of monoclonality in the urine, masses, and lymph nodes and minor clonality in the peripheral blood. These findings led to a diagnosis of EBL with minor clonality of B-cell in the peripheral blood.

Enzootic bovine leukosis (EBL) is a B-cell lymphoma caused by bovine leukemia virus (BLV) in cattle [1]. Neoplastic B-cells proliferate into the peripheral blood, several organs, and lymph nodes throughout the body, resulting in various clinical symptoms. Although lymphocytosis is a common finding of EBL [1], several EBL cases without lymphocytosis have been reported [5, 12, 14]. B-cell clonality test to detect monoclonal proliferation of B-cells has been reported [8] and is used as a diagnostic tool for EBL [2, 7]. Here we report a case of EBL in a Holstein-Friesian dairy cow without lymphocytosis and B-cell monoclonality in the peripheral blood.

A 4-year 9-month-old Holstein-Friesian dairy cow presented with anorexia. On day 1, a pelvic mass was palpated on rectal examination. Blood examination revealed anemia (red blood cell (RBC) count: 2.97 × 10⁶/μL, reference interval (RI): 5.10–7.60 × 10⁶/μL; hemoglobin concentration: 4.6 g/dL, RI: 8.5–12.2 g/dL; hematocrit: 14.1%, RI: 22–33%), but there was no lymphocytosis (lymphocyte count: 2,920/µL, RI: 1,600–5,600/µL) in the peripheral blood [3]. On day 6, the cow exhibited emaciation, anorexia, and a prolonged urination posture after urinating a small amount (Fig. 1). Urine were clear, and tumor cells were not observed in urine sediment smears. Swelling of left superficial cervical lymph nodes was observed, and fine needle aspiration (FNA) cytology revealed medium to large lymphoblast cells (70%) (Supplementary Fig. 1). Ultrasound examination revealed the presence of dilated renal calyces surrounded by perirenal anechoic areas in left kidney, suggesting hydronephrosis. Hematological examination revealed anemia (RBC count: 2.74 × 10⁶/μL; hemoglobin concentration: 4.9 g/dL; hematocrit: 13.3%) and no lymphocytosis (lymphocyte count: 4,182/μL). In peripheral blood smear examination, lymphocytes were mainly small, but several medium lymphoid cells were also detected (Supplementary Fig. 2). Serum biochemical analysis revealed increased levels of blood urea nitrogen (BUN: 49.1 mg/dL, RI: 10–25 mg/dL), creatinine (CRE: 3.0 mg/dL, RI: 0.4–1.0 mg/dL), total lactate dehydrogenase activity (LDH: 2,040 U/L, RI: 697–1,450 U/L), and thymidine kinase activity (TK: 15.2 U/L, RI: <5.4 U/L) [3, 6, 13]. The BLV copy number of DNA extracted from a peripheral blood sample was 1,118 copies/10 ng DNA, exceeding diagnostic criterion for BLV associated with tumor development (400 per 10 ng DNA) [11].

Fig. 1.

Prolonged urination posture after urinating a small amount on day 6. The hind limbs were spread, and the tail was raised.

The present case was clinically diagnosed with EBL, and the prognosis was determined to be poor. The cow was euthanized at the farm and transferred to the Veterinary Medical Center at The University of Tokyo for necropsy on day 11. At the necropsy, the left superficial cervical lymph node was swollen (20 × 10 × 10 cm) (Supplementary Fig. 3A), and a large mass (20 × 20 × 40 cm) in the pelvic cavity involving the rectum, bladder, and left ureter (Fig. 2) and several masses (2–15 cm) in the heart, abomasum, and gallbladder were observed (Supplementary Fig. 3B). The cut surface of the masses and the left superficial cervical lymph node were homogenous and white to grey in color. The bladder was dilated (30 × 45 × 10 cm) with the accumulation of cloudy urine (Supplementary Fig. 3C). The cytology examination of the urine sediment taken from the bladder revealed only debris of disintegrated cells. In the kidneys, caliectasis and white lesions were observed (Fig. 2). Histopathologically, medium to large neoplastic cells with a small amount of cytoplasm diffusely proliferated in the left superficial cervical lymph node, heart, abomasum, intestines, gallbladder, bladder, kidney, and the masses in the pelvic and abdominal cavities (Fig. 3). Immunohistochemically, tumor cells in the left superficial cervical lymph node were positive for CD79α (GeneTex, Irvine, CA, USA) and negative for CD3 (Dako, Santa Clara, CA, USA) (Fig. 4). Based on these findings, the present case was diagnosed as B-cell lymphoma.

Fig. 2.

Pelvic and abdominal cavity organs. A large pelvic mass (arrowheads) involved the rectum, bladder, and left ureter. The bladder was dilated. Caliectasis and white lesions were observed in the left kidney. Abnormalities were not observed in uterus and vagina. Bar=10 cm.

Fig. 3.

Histopathology of the left superficial cervical lymph node. Neoplastic cells were round and medium to large in size, with a small amount of cytoplasm. Hematoxylin and eosin stain. Bar=50 μm.

Fig. 4.

Immunohistochemistry of the left superficial cervical lymph node. Neoplastic cells were positive for CD79α and negative for CD3. (A) CD79α. (B) CD3 with Mayer’s hematoxylin counterstain. Bar=50 μm.

B-cell clonality was assessed by PCR amplification of immunoglobulin heavy chain gene V-D-J junctional variability [8] using DNA extracted from the peripheral blood, urine taken from spontaneous urination without centrifugation, urine taken from the bladder during necropsy without centrifugation, left superficial cervical lymph tissue obtained by FNA, left superficial cervical lymph, and a mass in the pelvic cavity. PCR was performed using HotStarTaq DNA Polymerase (QIAGEN, GmbH, Hilden, Germany) and a primer pair (BoVHF1 and BoVHR1). Genomic DNA from a typical EBL cow with enlarged lymph nodes and lymphocytosis was used as a positive control, and genomic DNA from cattle with inflammatory disease (mastitis) and distilled water were used as negative controls. PCR products were electrophoresed on a 2% agarose gel (Fig. 5). A smear and a single band were detected in the peripheral blood and other samples, respectively. PCR products were analyzed with a capillary electrophoresis device (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA) and a DNA 1000 Lab Chip kit (Agilent Technologies). A 152 bp peak exceeding the height of the marker was detected in the samples other than the peripheral blood (Fig. 6A), whereas a waveform with a 152 bp small peak not exceeding the height of the marker was detected in the peripheral blood sample (Fig. 6B). These results indicated B-cell monoclonality in the urine, enlarged lymph nodes, and masses, and minor clonality in the peripheral blood [15].

Fig. 5.

Results of agarose gel electrophoresis of PCR products of B-cell clonality test. Lane 1: peripheral blood; Lane 2: left superficial cervical lymph tissue obtained by FNA; Lane 3: urine taken from spontaneous urination without centrifugation; Lane 4: left superficial cervical lymph; Lane 5: mass in pelvic cavity; Lane 6: urine taken from the bladder during necropsy without centrifugation; Lane 7: typical EBL; Lane 8: endocarditis; Lane 9: distilled water; M: molecular weight marker.

Fig. 6.

Representative results of capillary electrophoresis of PCR products in B-cell clonality test. A 152 bp peak exceeding the height of the marker was detected using left superficial cervical lymph (A). A waveform with a 152 bp small peak not exceeding the height of the marker was detected using peripheral blood (B).

Swelling of superficial lymph nodes, pelvic masses, and lymphocytosis led to a suspension of EBL onset. In the present case, a pelvic mass was detected on rectal examination, and EBL was suspected on day 1; however, there was no lymphocytosis. On day 6, the left superficial cervical lymph node was enlarged, and FNA cytology of the enlarged lymph node revealed medium to large lymphoblast cells. Moreover, increased serum biochemical lymphoma markers (LDH and TK) [6, 13] and a high BLV copy number were detected [11]. Based on these findings, the present case was clinically diagnosed with EBL, despite no lymphocytosis. The pathological diagnosis of B-cell lymphoma supported the clinical diagnosis. Although several EBL cases without lymphocytosis have been reported [5, 12, 14], the proportion of EBL cattle without lymphocytosis has been unclear and will need to be clarified in future studies.

The B-cell monoclonality status with the 152 bp peak of enlarged lymph nodes and masses in the present case suggested neoplastic B-cells were proliferated and made up for most of the construction of B-cells in these tissues. On the other hand, B-cell clonality test indicated minor clonality, with the 152 bp small peak, in the peripheral blood. The 152 bp peak observed in B-cell clonality test of tumor tissue and peripheral blood showed the presence of tumor cells with same origin in both samples. The peak height in the B-cell clonality test indicates the proportion of clones. Therefore, the presence of a few neoplastic B-cells in the peripheral blood, although the proportion of tumor cells was not high enough to cause monoclonal proliferation. The number of lymphocytes was within the normal range in the precent case, while a case of EBL with B-cell monoclonality of peripheral blood lymphocytes in the absence of lymphocytosis has been reported [5]. Further investigation of the B-cell clonality status and the number of lymphocytes in cattle with EBL will be needed.

The kidney and ureter are predilection sites for bovine lymphoma [9]. A case of EBL clinically diagnosed based on the detection of atypical lymphocytes by urinary sediment examination has been reported [12]. The cow in the present case showed a prolonged urination posture after urinating a small amount on day 6; however, urine were clear, with no tumor cells detected in the smear examination of the urine sediment. Necropsy revealed tumor cell infiltration in the kidney and a large mass involving the rectum, bladder, and left ureter. Moreover, the bladder was dilated, and cloudy urine accumulated in the bladder. These findings suggest that the urination disorder might have been caused by the large pelvic mass, and that a small amount of urinary supernatant might be excreted in urination. The clonality test revealed B-cell monoclonality in both urine taken from spontaneous urination and that taken from the bladder during necropsy. In several tumors of human and dog, DNA of neoplastic cells was detected in urine [4, 10, 16, 17]. The urine samples might have contained DNA from broken tumor cells accumulated in the bladder. In cases of EBL with pelvic masses, PCR for B-cell clonality using urine in addition to urine sediment smears might improve the diagnostic accuracy.

Erythropoietin (EPO) is a glycoprotein hormone produced by the peritubular cells of the renal cortex, and inadequate erythropoietin production is one of the causes of anemia. In the present case, anemia was noted on days 1 and 6, and neoplastic lesions were observed in the kidneys on pathological examination. Decreased erythropoietin production due to destruction of the peritubular cells of the renal cortex by tumor cell infiltration might cause anemia in the present case.

In conclusion, the present case was diagnosed with EBL despite no lymphocytosis. The B-cell clonality test indicated minor clonality, with a few neoplastic cells present in the peripheral blood. Further investigation of the B-cell clonality status and the number of peripheral blood lymphocytes in cattle with EBL will be needed.

CONFLICTS OF INTEREST

The Laboratory of OSG Veterinary Science for Global Disease Management is an endowment laboratory, supported by a grant from OSG Corporation.