Search

Enzootic bovine leukosis (EBL), although eradicated in some European countries, is still the most common neoplastic disease of cattle, caused by the bovine leukemia virus (BLV). During the progression of EBL, BLV-infected cells clonally expand, and some of which result in tumor onset. The clonality of BLV-infected cells is generally evaluated with NGS or Sanger sequencing. Although these methods clearly distinguish EBL from non-EBL cases, the procedures are complex and not practical for routine veterinary diagnosis. In this study, we developed an amplified fragment length polymorphism (AFLP) analysis for BLV clonality assay (BLV-AFLP). This analysis uses restriction enzyme digestion to amplify the chimeric regions of BLV 3' linear transcribed region (LTR) and host genome through conventional polymerase chain reaction (PCR) and visualizes the results by gel-electrophoresis. The method was established using cattle samples representing different stages of the disease: BLV-uninfected, non-EBL, and EBL cattle. Non-EBL cattle showed smeared bands, indicating polyclonal proliferation, while EBL cattle showed distinct bands, indicating clonal expansion. The results of BLV-AFLP correlated well with those of previously reported methods, suggesting its efficacy in detecting clonal proliferation. The validation using blood samples of non-EBL cattle and tumor samples of EBL cattle confirmed that BLV-AFLP could effectively identify clonal proliferation in EBL samples. Moreover, the emergence of dominant clones in the tumor at later stages was successfully detected before EBL onset in some cattle, highlighting its sensitivity and potential for early detection. Overall, BLV-AFLP is suitable for practical use in the field, improving BLV management strategies and minimizing economic losses., Importance: Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5' end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

Bovine leukemia virus (BLV) is a causative agent of enzootic bovine lymphoma (EBL). BLV is prevalent worldwide, and ten genotypes have been classified based on the sequence of the envelope glycoprotein (gp51) gene. In this study, we present a simple and generally applicable PCR restriction fragment length polymorphism (PCR-RFLP) method to identify BLV genotypes. While the genotyping results obtained by previously described PCR-RFLP methods matched only 78.96% to the results of phylogenetic analysis, we demonstrated that our PCR-RFLP method can identify 90.4% of the sequences available in the database in silico. The method was validated with 20 BLV sequences from EBL tumor tissues and 3 BLV sequences from blood of BLV infected cattle, and was found to show high specificity. We utilized this method to determine genotypes of blood samples from 18 BLV seropositive cattle in Kanagawa and Niigata, as well as 12 EBL cattle in Chiba, Japan. Our analysis with the modified PCR-RFLP detected two genotypes, Genotypes 1 and 3. Genotype 1 was detected as the main genotype, while Genotype 3 was sporadically observed. This technique can be used as a reliable system for screening a large number of epidemiological samples.

Bovine leukemia virus (BLV) belongs to the genus Deltaretrovirus in the family Retroviridae, and is etiologically associated with Enzootic Bovine Leukosis (EBL). The majority of BLV-infected cattle remain asymptomatic throughout their productive lives, whereas approximately 5%-10% of infected cattle develop EBL. Data accumulated recently indicate that whole blood proviral load (PVL) levels of BLV-infected cattle could be an indicator of disease progression in the field. However, a few cross-sectional studies have been reported. Here, we prospectively evaluated 866 cattle enrolled between August 2015 and December 2015, and followed until November 2018, identifying 407 asymptomatic BLV-infected cattle. There were no significant differences in the median PVL level among the category of herd seroprevalence (p = 0.57), herd size (p = 0.19), nor among the category of past EBL history in the herd (p = 0.31). During the study period, 12 cattle developed EBL. The PVL levels of EBL cattle at the time of enrollment were significantly higher than that of cattle that did not progress to EBL (median, 90,695 vs 39,139 copies/10 5 cells, p = 0.0005). Moreover, the adjusted hazard ratio for the increase in PVL was 2.61 (95% CI, 1.51-4.53) as estimated by the Cox proportional hazards frailty model. These results indicate that a high PVL level is a significant risk factor for progression to EBL, and could potentially be used as an indicator for the identification of cattle to be culled from the herd long before the progression of EBL. This knowledge might be useful to design a strategy for decreasing economic loss from EBL or even eradicating it from herds., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: We had previously reported the short-term results of the aortic valve neocuspidization (AVNeo) procedure. We have now evaluated the midterm results with the longest follow-up of 118 months., Methods: From April 2007 through December 2015, 850 patients were treated with AVNeo using autologous pericardium. Medical records of these patients were retrospectively reviewed. The procedure was on the basis of independent tricuspid replacement using autologous pericardium. The distances between the commissures were measured with an original sizing device, the pericardial cusp was trimmed using an original template, and then sutured to the annulus., Results: There were 534 patients with aortic stenosis, 254 with aortic regurgitation, 61 with aortic stenoregurgitation, 19 with infective endocarditis, and 5 with a previous aortic valve procedure. Besides 596 patients with tricuspid aortic valve, 224 patients had bicuspid valve, 28 had unicuspid valve, and 2 had quadricuspid valve. There were 444 male and 406 female patients. The median age was 71 (range, 13-90) years old. Preoperative echocardiography revealed a peak pressure gradient average of 68.9 ± 36.3 mm Hg with aortic stenosis. Surgical annular diameter was 20.9 ± 3.3 mm. There was no conversion to a prosthetic valve replacement. There were 16 in-hospital mortalities. Postoperative echocardiography revealed a peak pressure gradient average of 19.5 ± 10.3 mm Hg 1 week after surgery and 15.2 ± 6.3 mm Hg 8 years after surgery. Fifteen patients needed reoperation (13 infective endocarditis, 1 break of thread, and 1 tear of cusp case). The mean follow-up period was 53.7 ± 28.2 months. Actuarial freedom from death, cumulative incidence of reoperation, and that of recurrent moderate aortic regurgitation or greater was 85.9%, 4.2%, and 7.3%, respectively, with the longest follow-up of 118 months., Conclusions: The midterm outcomes of AVNeo using autologous pericardium were satisfactory in 850 patients with various aortic valve diseases. However, further randomized, multicenter prospective studies are needed to confirm the results of the current study., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Background: To determine the feasibility of original aortic valve reconstruction (AVRec) for patients with aortic stenosis (AS), 416 consecutive cases were reviewed., Methods and Results: AVRecs for AS were performed for 416 patients from April 2007 through April 2013. All 416 patients were retrospectively reviewed. One hundred and fourteen patients had bicuspid valves and 16 had unicuspid valves. There were 182 men and 234 women. Mean age was 71.2±12.0 years old. On preoperative echocardiography, peak pressure gradient averaged 79.0±33.6 mmHg. Surgical annular diameter was 20.1±2.8 mm. The procedure is based on independent tricuspid replacement by autologous pericardium using original sizing apparatus and template. There was no conversion to prosthetic valve replacement. There were 8 in-hospital mortalities due to non-cardiac cause. On postoperative echocardiography, peak pressure gradient averaged 21.2±10.7 mmHg 1 week after surgery and 14.3±5.0 mmHg 5.5 years after surgery. Four reoperations were done for infective endocarditis. The other 412 patients had less than mild regurgitation. No thrombo-embolic events were recorded. The mean follow-up period was 25.2±17.5 months. Freedom from reoperation was 96.7% with 73-month follow-up., Conclusions: Medium-term results were excellent. Original AVRec was feasible for the patients with AS. Long-term data will be presented in the future.

Background: We performed original aortic valve reconstruction using autologous pericardium; the feasibility for elderly patients is reviewed., Methods: From April 2007 through September 2011, aortic valve reconstruction was carried out in 86 patients over the age of 80 years. Twenty-seven patients were male and 59 were female. Mean age was 82.9 ± 2.5 years. Seventy-two patients had aortic stenosis and 14 had aortic regurgitation. Mean preoperative surgical annular diameter was 19.5 ± 2.5 mm. There were 80 (90.7%) cases of small aortic annulus. Mean preoperative logistic EuroSCORE was 22.9 ± 15.8., Results: Isolated aortic valve reconstructions were performed in 51 patients. Concomitant procedures included coronary artery bypass grafting in 6, hemiarch aortic replacements in 6, 9 maze procedures, and some combinations. No conversion to valve replacement was required. Mean follow-up was 1243 days. There were 3 hospital deaths due to noncardiac causes. No reoperation was needed. Survival at 56 months was 87.0%. No thromboembolic event occurred. Echocardiography 3.5 years after surgery revealed an average peak pressure gradient of 14.6 ± 3.8 mm Hg. No moderate or severe regurgitation was recorded., Conclusions: Aortic valve reconstruction is feasible for patients older than 80 years, resulting in good hemodynamics and a better quality of life, without anticoagulation., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Objective: We have performed an original aortic valve reconstruction using autologous pericardium. The feasibility for patients aged less than 60 years is reviewed., Methods: From April 2007 to April 2013, aortic valve reconstruction was performed in 108 patients aged less than 60 years. A total of 51 patients had aortic stenosis, 7 patients had annuloaortic ectasia, 7 patients had infective endocarditis, and 43 patients had aortic regurgitation. Fifty-seven patients had bicuspid valves, and 11 patients had unicuspid valves. There were 75 male and 33 female patients, with a mean age of 47.8 ± 11.2 years. Preoperative echocardiography showed an average peak pressure gradient of 86.1 ± 35.1 mm Hg with aortic stenosis. The surgical procedure is based on the independent tricuspid replacement using autologous pericardium. First, the distance between the commissures is measured using an original sizing apparatus, and then the pericardial cusp is trimmed using an original template and sutured to the annulus., Results: There was no conversion to prosthetic valve replacement. There were no in-hospital mortalities. Postoperative echocardiography showed an average peak pressure gradient of 14.8 ± 7.8 mm Hg 1 week after surgery and 12.8 ± 3.1 mm Hg 4 years after surgery. One patient required reoperation because of infective endocarditis. The other 107 patients showed less than mild aortic regurgitation. No thromboembolic events were recorded. The mean follow-up period was 34.2 ± 15.7 months. Freedom from reoperation was 98.9% with 76 months of follow-up., Conclusions: Original aortic valve reconstruction was feasible for patients aged less than 60 years. Long-term data will be disclosed in the future., (Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Background: This study describes the surgical treatment of bicuspid aortic valve with original aortic valve reconstruction., Methods and Results: Aortic valve reconstruction was done in 102 patients with bicuspid aortic valve from April 2007 through September 2011. Thirty-four patients with ascending aortic diameter >45 mm underwent hemi-arch aortic replacement concomitantly. Seventy-seven patients had aortic stenosis, and 25 had aortic regurgitation (AR). Mean age was 63.7±10.0 years old. There were 55 men and 47 women. Harvested pericardium is treated with 0.6% glutaraldehyde solution. The distance between commissures is measured with an original sizing instrument. For bicuspid valve with raphe, the raphe is considered as a commissure in order to measure the distance between each commissure. Without a raphe, we create a new annular margin and commissure using coronary ostium and the sizing instrument as a guide. Then, pericardium is trimmed with original template. Three cusps are sutured independently. The preoperative averaged peak pressure gradient of 71.1±39.0 mmHg was decreased to 16.2±8.8, 13.3±6.0, and 13.9±5.6 mmHg, respectively 1 week, 1 year, and 3 years after operation. AR was trivial. One reoperation was recorded. Mean follow-up was 733 days. There were 5 late mortalities. No thromboembolic event was recorded., Conclusions: Medium-term results were excellent. Tricuspidization gave good opening and closure of aortic valve with excellent hemodynamics.