Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA.

Quantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.

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

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs.

Seroprevalence of bovine leukemia virus in dairy cattle in Argentina: comparison of sensitivity and specificity of different detection methods.

Bovine leukemia virus (BLV) is a retrovirus that induces a chronic infection in cattle, which develop in three possible pathological forms: asymptomatic course, persistent lymphocytosis (PL) and lymphosarcoma. Once infected, cattle remain virus carriers for life and start to show a serological reaction within a few weeks after infection. Eradication and control of the disease is based on early diagnostic and segregation of the carriers. The agar gel immunodiffusion (AGID) test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA) has replaced the AGID for large scale testing. Although Argentina has over 60 million cattle population, no nationwide studies have been conducted yet to determine the prevalence of the infection. To estimate the rate of BLV infection in dairy cattle in Argentina, a survey for specific antibodies in >10,000 serum samples from animals over 18 months old, belonging to 363 different herds from the largest dairy production areas of the country, was carried out in our laboratory, along 1999. For this purpose, we developed an ELISA to detect serum antibodies against the BLV virus. The cut-off of the ELISA was established over 339 serum samples, using polymerase chain reaction and southern blot (PCR-SB) as confirmatory test. The sensitivity and specificity of the ELISA was of 97.2 and 97.5%, respectively, while the local official AGID test showed a sensitivity of 79.7% and specificity of 99.0%. To know the seroprevalence of BLV on dairy herds, and also the incidence of the infection within the herd, the serological survey was based on individual serum samples. The results show that the prevalence of infected individuals is of 32.85%, while the percentage of infected herds, harboring one or more infected animals, is of 84%. These results indicate a medium level of seropositive animals when taken individually, but a high prevalence of infected farms, which has been notoriously increased in the last 15 years as shown when compared with previous data from particular geographic areas, indicating that BLV constitutes a serious sanitary problem for dairy producers in Argentina. They also indicate the poor sensitivity of the official AGID test used in the country.