Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction

Petersen, Marcos Iván; Álvarez, Irene; Trono, K.; Jaworski, Juan Pablo

doi:10.3168/jds.2017-14253

Cited by 20 publications

(25 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The low proviral loads in milk might be explained by the presence of numerous components in milk (i.e., proteases, complex polysaccharides, lipids, and Ca 2+ ions) that can interfere with DNA extraction and qPCR analysis, thereby, decreasing the detection sensitivity compared to that in the blood. Indeed, interference with PCR amplification was previously demonstrated when the limit of detection of BLV qPCR was assessed in the presence of the BLV provirus in a milk matrix [16]. Another explanation for the differences in proviral loads could be that less BLV-infected cells containing integrated BLV provirus are present in milk than in blood.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, it was previously reported that BLV-infected cells were present in the milk and colostrum of BLV-positive dams, because inoculation of lambs with milk or viable milk cells from 24 dairy cattle naturally infected with BLV resulted in the detection of infectious virus in the milk of 17 cows [13]. Recently, BLV provirus was detected in field samples of milk and colostrum [14–16]. However, the resistance of calves to milk-borne infection can be attributed to virus-neutralizing antibodies, which all calves nursed on BLV-positive dams acquire through the colostrum and retain in their serum for as long as 6 months [17, 18].…”

Section: Introductionmentioning

confidence: 99%

“…Genomic DNA can be extracted from various sources, including whole blood, milk, semen, saliva, nasal secretions, and several organs [14–16, 29–37]. We previously developed a highly specific, accurate, and sensitive method for quantifying proviral loads for both known and novel BLV variants in animals naturally infected with BLV [31, 38–40], based on the use of coordination of common motifs (CoCoMo) primers.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Watanuki

Takeshima

Borjigin

et al. 2019

Vet Res

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Watanuki

Takeshima

Borjigin

et al. 2019

Vet Res

View full text Add to dashboard Cite

show abstract

“…PVL was quantified by a real-time quantitative PCR (qPCR), as previously reported [10,11,12]. Briefly, the SybrGreen chemistry was used, amplifying a region of 120 bp of the pol gene and 500 ng of DNA template.…”

Section: Methodsmentioning

confidence: 99%

Detection of Bovine Leukemia Virus RNA in Blood Samples of Naturally Infected Dairy Cattle

Álvarez

Porta

Trono

2019

Veterinary Sciences

View full text Add to dashboard Cite

The viral expression in vivo, in bovine leukemia virus (BLV)-infected cattle, is considered to be restricted to extremely low levels, and the mitosis of infected B lymphocytes is regarded as the main mode of virus persistence within the infected host. In this study, the presence of BLV RNA in whole blood from seven asymptomatic cows naturally infected with BLV during one year, including a complete milking cycle and two delivery time points, was investigated by nested-PCR using the oligonucleotides complementary to the tax and pol gene. BLV RNA was detected in four cows at different time points, especially in high blood proviral load cows and around delivery time. This study describes the detection of free BLV RNA in blood from BLV-infected asymptomatic cows. The results obtained suggest the occurrence of persistent low-level expression of the tax and pol genes that could be a result of viral reactivation, within the asymptomatic period. This finding may be important in the pathogenesis of BLV infection, associated with the delivery period.

show abstract

“…The quantification of BLV proviral genome is an important diagnostic tool for BLV infection. Compared with serological assays, real-time PCR has high sensitivity and can act as a confirmatory test for the clarification of inconclusive or discordant serological test results (Petersen et al, 2018). However, BLV provirus cannot be detected by real-time PCR in some BLV-infected cattle (not published).…”

Section: Discussionmentioning

confidence: 99%

Establishment of a novel diagnostic test for Bovine leukaemia virus infection using direct filter PCR

Daous

Mitoma

Elhanafy

et al. 2020

Transbound Emerg Dis

View full text Add to dashboard Cite

Enzootic bovine leucosis (EBL) is a neoplastic disease caused by Bovine leukaemia virus (BLV) (Bartlett et al., 2014). BLV infection occurs in three possible stages: the aleukaemic stage, persistent lymphocytosis (PL) stage and EBL stage (Dequiedt et al., 1999). It is estimated that approximately 30% of BLV-infected cattle are in the PL stage, and fewer than 5% of those develop fatal B-cell lymphosarcoma (Rodríguez et al., 2011). This disease has an economic impact because it causes direct economic losses by reducing milk production and fertility, and through cattle condemnation and death (Kale et al., 2007). A great amount of efforts was directed to BLV elimination during the period 1993-2010 (EFSA Panel on Animal Health and Welfare (AHAW) et al., 2017) in the European Union (EU). As a result, most of its member states have officially declared EBL-free. In Japan, on the other hand, the nationwide survey of BLV infection from 2009 to 2011 showed that the prevalence of BLV infection in animal level was 40.9%

show abstract

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction

Cited by 20 publications

References 17 publications

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

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

Detection of Bovine Leukemia Virus RNA in Blood Samples of Naturally Infected Dairy Cattle

Establishment of a novel diagnostic test for Bovine leukaemia virus infection using direct filter PCR

Contact Info

Product

Resources

About