Sample collection, extraction of genomic DNA and diagnosis

Blood samples were obtained three times (Stages I, II, and III) from a naturally infected Holstein dairy cow in a breeding farm located in Chiba Prefecture, Japan (Table 1). Serum samples were also obtained from the same cow.

BLV infection was evaluated using BLV-CoCoMo-qPCR-2 (RIKEN Genesis, Kanagawa, Japan) with THUNDERBIRD Probe qPCR Mix (TOYOBO Co., Ltd., Osaka, Japan) [35–37], and an anti-BLV antibody enzyme-linked immunosorbent assay (ELISA) kit (JNC Inc., Tokyo, Japan). The subclinical stage of BLV infection was diagnosed according to the lymphocyte count (cells/µL) and age of each cow (≤ 7500 = normal and ≥ 9500 = lymphocytosis for cows aged 3–4 years; ≤ 6500 = normal and ≥ 8500 = lymphocytosis for cows aged ≥ 4–5 years) [16]. BLV-infected but clinically and hematologically normal cows were defined as asymptomatic cattle, whereas BLV-infected but clinically normal cattle showing an increase in the number of apparently normal B lymphocytes were defined as PL cows. Subsequently, lymphoma in cattle was diagnosed based on gross and histological observations. WBC and lymphocytes was measured using an automated veterinary hematology analyzer (MEK-6550 Celltac α, Nihon Kohden, Tokyo, Japan).

This study was approved by the animal ethics committee and the animal care and use of the RIKEN Animal Experiments Committee (Approval Number H29-2-104).

Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA)-treated blood samples according to the protocol provided with Wizard Genomic DNA Purification Kit (Promega Corporation, Tokyo, Japan).