Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis

Abstract

Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.

Keywords: bovine leukemia; early onset EBL; expression patterns of B-cell markers; immunophenotyping.

FIG 1

The relationship between population count in cell marker expression, B-cell clonality, and bovine leukemia virus (BLV) provirus loads. (A) The percentages of single (tumorigenic phenotype) or multiple (normal phenotype) cell populations for each cell marker were compared between high and low B-cell clonality. In all 176 samples from 50 cattle, 80 samples were of high clonality (H), 63 were of low clonality (L), and 33 were of unclear results or there were no data from flow cytometry analysis. (B) The relationship between B-cell clonality and BLV provirus loads. In all 176 samples from 50 cattle, 84 samples were of high clonality (H), 55 were of low clonality (L), and 37 were of unclear results or there were no data of provirus loads. The 50 cattle include 26 with classic enzootic bovine leukosis (cEBL) (H, n = 72; L, n = 15) and 24 non-cEBL cattle (H, n = 12; L, n = 40). *, P < 0.05 by Kruskal-Wallis test followed by Steel-Dwass test; n.s., not significant.

FIG 2

Breed and age of the cattle with lymphomas. The percentages and numbers of each type of lymphoma were compared according to the breed (left) and age (right) of the cattle. JB, Japanese Black; HO, Holstein. Numbers on the x axis on the right indicate age in years.

FIG 3

Lack of lymphocytosis in cattle with classic enzootic bovine leukosis (cEBL). (A) The numbers of lymphocytes in peripheral blood samples from cEBL cattle are shown (n = 20). Open bars indicate that the lymphocyte count was <10,000 cells per 1 μl of blood. (B) Correlation between the numbers of whole blood cells and lymphocytes in peripheral blood samples from cEBL cattle (n = 20).

FIG 4

Immunophenotyping based on expression pattern of cell markers between three B-cell lymphomas and healthy controls. (A) The expression levels of six cell markers in samples with classic enzootic bovine leukosis ([cEBL] n = 42), polyclonal EBL ([pEBL] n = 15), B-cell-type sporadic bovine leukosis ([B-SBL] n = 7), and healthy controls (n = 17) are shown as box-and-whisker plots. Each box indicates the median and lower and upper quartiles and whiskers indicate lower and upper extremes. The “x” marks indicate the averages, and dots represent outliers that are much greater than normal or much less than normal. *, P < 0.05 by Kruskal-Wallis test followed by Steel-Dwass test. (B) Dual expressions of IgM, WC4, and CD21 in cEBL, pEBL, and B-SBL. Each axis indicates the percentages of positive cells, and the bubble size indicates the number of samples which showed identical expression patterns. (C) Triple expression of IgM, WC4, and CD21 in cEBL, pEBL, and B-SBL. +, cell marker expression ≥ 50%; −, cell-marker expression < 50%.

FIG 5

Discriminant analysis of classic enzootic bovine leukosis (cEBL), polyclonal EBL (pEBL), B-cell-type sporadic bovine leukosis (B-SBL), and healthy controls. (A) Discriminant scores giving a classification performance of B-cell lymphomas and controls based on a linear discriminant analysis with the expression of six cell markers: CD5, IgM, WC4, CD21, CD79a, and CD3. (B) Scatter plots of multiple discriminant analyses to visualize the clustering of cell marker expression from each B-cell lymphoma and from healthy controls. First, second, and third linear discriminants are shown as LD1, LD2, and LD3, respectively. cEBL, n = 41; pEBL, n = 13; B-SBL, n = 7; healthy control, n = 17.