Background

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), a form of B cell lymphoma in cattle, which is closely related to human T-cell leukemia viruses (HTLVs) [1]. BLV has spread worldwide and causes serious problems in the cattle industry [2]. Transmission of BLV within a herd can occur primarily through contact with bodily fluids containing infected lymphocytes via blood-sucking insects or through iatrogenic procedures, including repeated use of individual needles, syringes, rectal palpation gloves, and dehorners [3]. Initially, BLV infects various cell populations, but only BLV-infected B cells can induce the mono- or oligo-clonal expansion of origin of B-cells after a long latency period [1]. Eventually, BLV-induced B-cell lymphoma can occur in different organs and tissues, leading to a series of defects that cause mortality in the animal [3]. The majority of BLV-infected cattle are asymptomatic carriers of the virus, but less than 5% of infected cattle develop lymphoma [1]. This means that most infected cattle remain healthy, produce optimal amounts of milk, and are therefore, not culled from the herd. Therefore, BLV-infected B cells circulate through the blood of infected cattle and are present in beef [4], and infectious BLV-infected cell are also present in the milk of infected dairy cows [5,6,7]. This can be a means of transmission from cows to human. However, whether BLV is transmissible in humans remains unclear.

BLV naturally infects cattle, water buffalo, yaks [8] and alpaca [9,10,11], but can experimentally infect various other species, including sheep [12,13,14,15], rats [16], rabbits [16,17,18], pigs [19], chickens [19, 20] and goats [21] as well as several cell lines including human cells such as Hela cells and kidney 293T cells [22, 23]. Human cells of neural origin are also highly susceptible to BLV infection [16].

A few studies on the development of new and more sensitive methods have revealed re-emerging concern regarding the possible connection of BLV and human disease. An initial study by Buehring et al. reported the detection of antibodies against BLV p24 protein in 74% of the tested human sera [24]. A decade later, the same group also detected the BLV proviral gene tax in 44% (97/219) of human breast cancer tissue samples using in situ polymerase chain reaction (PCR), and 6% (12/215) of these samples showed the presence of viral p24 protein by immunohistochemistry [25]. Since then, either the BLV provirus (gag, env, tax, long terminal repeat (LTR)) and/or antibodies reacting to BLV antigens (p24 and gp51) have been detected in a wide range of geographical regions such as the United States [26,27,28,29], Australia [30, 31], Columbia [32], Brazil [29, 33] and Iran [34], in human breast tissue (healthy, benign, pre-malignant, or malignant), as well as in human blood and serum samples. These results indicate a high odds ratio of association between the presence of BLV DNA and breast cancer through screening the presence of BLV in the breast tissue of women with cancer and that of women without cancer using in situ PCR [26, 27, 29, 32]. BLV proviral DNA has also been detected in human squamous cell lung carcinoma [BLV proviral detection by PCR amplification with short proviral region

All samples were tested for presence of the BLV proviral genome using high-sensitive PCR method described previously [47], using the TaqMan Gene Expression Master Mix (Applied biosystems, Tokyo, Japan) with moderate modifications. Six proviral gene fragments, named LTR, gag, pol, env, tax, and tax-3`LTR were amplified using primers listed in Additional file 1: Table S1. The reaction mixture (20 μL/sample) contained 500 ng of sample DNA (The amount of FLK-BLV template DNA depend on the BLV copy numbers), 0.2 mM each primer and 1X TaqMan Gene Expression Master Mix. The PCR conditions were as follows: 95 °C for 5 min, followed by 60 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s (extension at 72 °C for 50 s for tax-3′LTR), followed by post-extension (1 cycle of 72 °C for 4 min) and a final hold at 4 °C. PCR reaction of each sample was performed in triplicate. After amplification, the PCR products were separated by electrophoresis on a 3.0% agarose gel based on size differences. Samples were considered positive for the BLV proviral genome region only if PCR results were obtained at least twice from three different reactions for each target gene.

Throughout the laboratory work with initial samples and DNA, special precautions were taken to prevent cross-contamination among test samples and positive control. DNA was extracted in a clean area, DNA-free room, using a special hood with UV light, along with nucleic acid decontamination solutions for preparing the PCR mixture as well as the addition of DNA to the reaction mix; all work with the positive control DNA, including DNA dilution based on copy number and addition of positive control into the PCR tube, was performed in another room.

ELISA for anti-BLV p24 antibody

The indirect BLV p24 ELISA was performed based on previously described [57]. ELISA plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) were coated with 200 ng/well recombinant BLV capsid p24 antigen diluted in 100 μL in 0.1 M NaHCO3 coating buffer (pH 9.0). After overnight incubation at 4 °C, coating buffer was removed and wells were washed for four times for 1 min per wash, with 300 μL/well/wash phosphate-buffered saline (PBS)-T buffer. Human serum specimen diluted 1:50 in sample dilution buffer (7.20 mM Na2HPO4 12H2O, 2.82 mM NaH2PO4 2H2O, 150.24 mM NaCl, 1% tween 20, and 5% skim milk) were incubated with antigens for 1 h at 37 °C, as primary antibodies. The plates were then washed and incubated at 37 °C for 30 min with HRP-conjugated goat anti-human IgG (diluted as 1/1000) (Abcam PIC., Tokyo, Japan) and goat anti-human IgM (diluted as 1/10,000) (Abcam PIC.), used as secondary antibodies. Finally, 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific, Rockford, CA) was applied to the plates and reacted with the test sample for 10 min in dark. The reaction was stopped adding 1 N H2SO4. OD values were measured at 450 nm with the correction at 690 nm using a EnSight™ multimode plate reader (PerkinElmer Inc., Waltham, MA). The plates were blanked on wells containing only the sample dilution buffer. All samples were run in triplicate. During each assay, to check the validity of the experiment procedure, two control systems were used: (i) Samples from two BLV-infected cattle and one cattle with no BLV infection, were used as positive and negative controls, respectively, followed by incubation with HRP-conjugated rabbit anti-bovine IgG (Thermo Fisher Scientific, Waltham, MA) (diluted as 1/2000), as the second antibody. (ii) To validate whether the human secondary antibodies (HRP-conjugated goat anti-human IgG and IgM) are active, we selected HTLV I/II antibody ELISA kit (MyBioScource Inc., San Diego, CA) and performed ELISA with replacement of secondary antibody in the kit with HRP-conjugated goat anti-human IgG and IgM.

ELISA for anti-BLV gp51 antibody

Two antibody isoforms (IgG and IgM) in human serum were assessed using a commercially available BLV indirect gp51 ELISA kit (JNC, Tokyo, Japan), according to the manufacturer’s instructions. Human serum specimens, diluted in 1:50 with the sample dilution buffer in the kit, were used as primary antibodies, followed by HRP-conjugated goat anti-human IgG (diluted as 1/1000) (Abcam PIC.) or goat anti-human IgM (diluted as 1/10,000) (Abcam PIC.) the secondary antibody. All samples were run triplicate. During each assay, to check the validity of the experiment procedure, we used two controls, as indicated in ELISA for anti-BLV p24 antibody.

Western blotting assay

FLK-BLV cells and BLV-free FLK cells were lysed for 30 min on ice in 20 mM Tris–HCl (pH 7.4), 300 mM NaCl, 2 mM ethylenediaminetetraacetic acid, and 2% NP40 supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lysates were mixed with SDS buffer and boiled for 5 min. Protein concentrations were determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). The proteins were then diluted in PBS to a final concentration of 10 ug/mL. Equal amounts of protein were electrophoresed by 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a Trans-Blot Turbo apparatus (Bio-Rad, Irvine, CA). After transfer, the membranes were incubated with blocking buffer with 8% skim milk for 30 min at room temperature and then incubated with human serums were diluted 1:50 with PBS with 5% skim milk, overnight at 4 °C. After washing, the membranes were incubated with HRP-conjugated goat anti-human IgG and IgM (1:100; Abcam PIC.) for 1 h. Sera from cattle infected with EBL and from uninfected cattle were used as positive and negative control, respectively, followed by incubation with rabbit anti-bovine IgG (1:4000; Thermo Fisher Scientific). The signals were visualized using SuperSignal™ West Femto Meximum Sensitivity Substrate (Thermo Fisher Scientific). Images were acquired using Chemiluminescent Imaging system (ATTO Corporation, Tokyo, Japan). PBS was used in place of primary antibody to adjust for any non-specific binding of the secondary antibody. Each human serum samples were tested twice on different days.