Visualizing bovine leukemia virus (BLV)-infected cells and measuring BLV proviral loads in the milk of BLV seropositive dams

Abstract

Bovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission.

Figure 1

Scatter diagrams showing the quantity and quality of DNA in blood and milk. DNA samples were obtained from 2 BLV-negative cattle in Farm 3, 43 BLV-infected cattle without lymphoma in Farms 2–6, and 3 BLV-infected cattle with lymphoma in Farm 1. A The average quantities of genomic DNA in blood and milk samples were 7.9 µg and 6.7 µg, respectively, as determined with a NanoDrop spectrophotometer ND-1000. B The average A260/A280 ratios of genomic DNA in blood and milk were 1.88 and 1.87, respectively, as determined using a NanoDrop spectrophotometer ND-1000. C The threshold cycle values of the blood and milk samples were 22.12 and 22.37, respectively, as indicated with the red bold lines.

Figure 2

Correlation between the BLV proviral loads in blood and milk. The proviral loads in milk and blood samples from 47 cattle in Farms 1–6 were measured by BLV-CoCoMo-qPCR-2 and compared after normalization to the levels found in 10⁵ cells. The bold line represents the approximate curve (r = correlation coefficient) and p value is indicated.

Figure 3

DNA-sequence alignment of the partial BLV gp51 env gene region isolated from blood and milk. The FLK-BLV sequence (EF600696) is shown as reference at the top of the sequence alignment. DNA fragments derived from blood and milk samples of two BLV-infected cattle with lymphoma (cows A1 and A2) and a BLV-infected cow without lymphoma (cow K12). The nucleotide (nt) sequences were compared with the FLK-BLV of gp51 env region (5078 nt–5599 nt). Sequence identity with the FLK-BLV sequence is indicated with dots.

Figure 4

BLV infectivity of milk cells from a BLV-infected cow with lymphoma (A1) via conventional LuSIA. A Milk cells were isolated from 100 mL milk sample and were resuspended in 1 mL of DMEM. Then milk cells (1 × 10⁵) were cultured for 4 days with CC81-GREMG cells. Subsequently, the cells were fixed in 3.6% formaldehyde/PBS with Hoechst 33342. The fluorescent syncytia were observed using an EVOS2 fluorescence microscope. FLK-BLV cells, which were productively infected with BLV, were used as the positive control. Mock-treated cells were used as the negative control. B Low-magnification image of the white square in subpanel A1. The scale bars (white bars) signify 275 µm.

Figure 5

Infectivity of milk cells from BLV-infected, healthy cows with our improved LuSIA using CC81-GREMG cells. A The proviral loads in blood and milk, and the improved LuSIA results of milk cells for 6 BLV-infected cattle without lymphoma (cows N1, K11, N5, K8, GDF211 and GDF349). B Typical visualization of the infectivity of milk cells from selected cows N1, K11, and N5 among 6 BLV-infected dam tested in this study using our improved LuSIA protocol. The cells remaining in the supernatants were pelleted at 800 × g (cows N1 and K11) or 1000×g (cow N5) for 30 min. Milk cells (1 × 10⁵) were cultured with 1 × 10⁴ CC81-GREMG cells with (+) or without (−) 1 µg/mL of PWM. After 5 days of co-culturing CC81-GREMG cells with milk cells, the cells were fixed in 3.6% formaldehyde/PBS with Hoechst 33342. Fluorescent syncytia were observed using an EVOS2 fluorescence microscope. +, more than two syncytia were observed in the milk cells. FLK-BLV cells, which were productively infected with BLV, were used as the positive control. The scale bars (white and yellow bar) signify 100 µm or 650 µm.