Irene Alvarez

Preventive and Therapeutic Strategies for Bovine Leukemia Virus: Lessons for HTLV

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle, segregation of BLV-free animals and vaccination. Although having been instrumental in regions such as the EU, these strategies were unsuccessful elsewhere mainly due to economic costs, management restrictions and lack of an efficient vaccine. This review, which summarizes the different attempts previously developed to decrease seroprevalence of BLV, may be informative for management of HTLV-1 infection. We also propose a new approach based on competitive infection with virus deletants aiming at reducing proviral loads.

Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.

Natural progression of Bovine Leukemia Virus infection in Argentinean dairy cattle

We describe the progression of Bovine Leukemia Virus (BLV) infection from birth until the first lactation in 61 animals from a typical large dairy herd of Argentina, with more than 85% of prevalence. The purpose was to identify potential points to effectively break the BLV cycle of transmission in our dairy productive system. We detected early infection in 11.47% of newborn calves by nested PCR. From birth to 12 months, no evidence of new infections was observed. After 12 months of age, the detection of new reactors increased slowly with time, from 15.09% at 15 months to 24% at 27 months. After that, the number of reactors increased rapidly up to 40% and 60.76% at 30 and 36 months, respectively. This last 9-month period coincided with parturition and the entry into the milking herd. Real-time PCR showed that more than 75% of adult animals had low peripheral-blood proviral load. Complementary, all infected animals showed low levels of provirus in milk and colostrum. The most important finding was that even when management procedures to prevent BLV iatrogenic transmission were followed, no significant change was observed in the prevalence after three years, strongly suggesting that other way/s of transmission play a key role under natural conditions. This study showed an interesting baseline to draw an alternative approach based on selective segregation according to the peripheral-blood proviral load as a potential indicator of risk transmission, and as an alternative to classical control measures.

Vaccination against δ−Retroviruses: The Bovine Leukemia Virus Paradigm

Bovine leukemia virus (BLV) and human T-lymphotropic virus type 1 (HTLV-1) are closely related δ-retroviruses that induce hematological diseases. HTLV-1 infects about 15 million people worldwide, mainly in subtropical areas. HTLV-1 induces a wide spectrum of diseases (e.g., HTLV-associated myelopathy/tropical spastic paraparesis) and leukemia/lymphoma (adult T-cell leukemia). Bovine leukemia virus is a major pathogen of cattle, causing important economic losses due to a reduction in production, export limitations and lymphoma-associated death. In the absence of satisfactory treatment for these diseases and besides the prevention of transmission, the best option to reduce the prevalence of δ-retroviruses is vaccination. Here, we provide an overview of the different vaccination strategies in the BLV model and outline key parameters required for vaccine efficacy.

Bovine leukemia virus p24 antibodies reflect blood proviral load

BackgroundBovine leukemia virus (BLV) is worldwide distributed and highly endemic in Argentina. Among the strategies to prevent BLV dissemination, a control plan based on the selective segregation of animals according to their proviral load (PVL) is promising for our dairy productive system. The objective of this work was to study the relationship between the blood PVL and the antibody level, in order to identify whether the individual humoral response, i.e. the anti-p24 or anti-whole-BLV particle, could be used as a marker of the blood level of infection and thus help to recruit animals that may pose a lower risk of dissemination under natural conditions.ResultsThe prevalence of p24 antibodies on the 15 farms studied was over 66%. The prevalence of p24 and whole-BLV antibodies and PVL quantification were analyzed in all the samples (n = 196) taken from herds T1 and 51. ROC analysis showed a higher AUC for p24 antibodies than whole-BLV antibodies (Zreactivity: 3.55, P < 0.001; Ztiter: 2.88, P < 0.01), and as consequence a better performance to predict the proviral load status in herd 51. No significant differences were found between the performance of p24 and whole-BLV antibodies in herd T1. A significant positive correlation was observed between PVL values and p24 antibody reactivity in both farms (r T1 = 0.7, P < 0.001, r 51 = 0.71, P < 0.0001). The analysis was extended to the whole number of weak p24 antibody reactors (n = 311) of the other 13 farms. The mean of high PVL reactors within weak p24 reactors was 17.38% (SD = 8.92). In 5/15 farms, the number of weak p24 reactors with high PVL was lower than 10%.ConclusionsWe found that the humoral response reflected the level of in vivo infection, and may therefore have useful epidemiological applications. Whereas the quantitative evaluation of blood proviral load using real-time PCR is expensive and technically demanding, the measurement of antibodies in blood by ELISA is relatively straightforward and could therefore constitute a cost-effective tool in a BLV control intervention strategy, especially in highly infected herds such as Argentinean dairy ones.

Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA.

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was expressed in the Escherichia coli expression system, purified in a nickel charged resin and used as antigen in the ELISA test. A cut-off point was established considering the distribution of reactivity values obtained by rp24-ELISA on a set of 555 field serum samples that were stated as double positive (DP) or double negative (DN) by the combination of commercial agar gel immunodiffusion (AGID) assay and gp51-ELISA tests results. The rp24-ELISA showed good concordance with the agar gel immunodiffusion assay, when 710 serum samples were analyzed. In addition, rp24-ELISA demonstrated to be a precise assay with good repeatability and reproducibility and a better analytical sensitivity than AGID. From 67 discordant rp24-ELISA-AGID sera, 4 negative reactors were from the DP group, 28 positive samples were from the DN group and 35 positive samples were stated as negative by AGID but positive by the gp51-ELISA. Samples from this last subgroup were sent to an OIE reference laboratory where 28 samples were stated as negative, 5 samples were stated as positive and 2 as inconclusive. Complete concordance with blind previous results from an international proficiency test panel confirmed the capability of the assay to discriminate between infected and non-infected animals. In conclusion, the rp24-ELISA developed and standardized demonstrated to have good analytical characteristics to be considered for screening of BLV.

Immunochromatographic lateral flow test for detection of antibodies to Equine infectious anemia virus

The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for comparative diagnostic sensitivity (98.3%), diagnostic specificity (87.4%) and concordance (92.4%) were similar to those reported for other ICLF tests for animal infectious diseases. Very good repeatability and reproducibility, as well as a total agreement with blind previous results from three proficiency test panels, were obtained, thus indicating that rp26-ICLF is a precise test. The end point of the twofold serial dilution of serum samples was the same as, and even better than, the AGID test, thus demonstrating the same analytical sensitivity as that of the reference method for EIA diagnosis. No cross-reactivity was observed when serum samples from horses with other infectious diseases were analyzed. rp26-ICLF proved to be a precise and rapid test suitable for field screening in veterinary practice, since minimal equipment and operator expertise are required. However, further research should be carried out to increase the level of sensitivity.

Characterization of colostrum from dams of BLV endemic dairy herds.

Bovine Leukemia Virus (BLV) is endemic in Argentina, where the individual prevalence is higher than 80% in dairy farms. The aim of this work was to find preliminary evidence to know if the high level of infection of the dam would implicate a higher challenge to her own offspring. We collected 65 sets of samples consisting of dams blood and colostrum from two heavily infected dairy farms, and investigated the correlation between the dams blood proviral load and the presence of provirus in colostrum. We also described the dual antibody/provirus profile in the colostrum. Provirus was detected in 69.23% of the colostrum samples, mostly from dams with a high proviral load, 36/45 (80%). Colostrum proviral load was significantly higher in dams with high blood proviral load (p<0.0001). Provirus was detected in colostrum samples all along the antibody distribution, even in those with a low amount of antibodies. These results show that even when high blood proviral load dams offer higher levels of infected cells to their offspring through colostrum they also offer higher levels of protection of antibodies. On the contrary, low blood proviral load dams also offer infected cells but a poor content of antibodies, suggesting that these animals could play an important role in the epidemiological cycle of transmission.

Evaluation of total white blood cell count as a marker for proviral load of bovine leukemia virus in dairy cattle from herds with a high seroprevalence of antibodies against bovine leukemia virus

OBJECTIVE To determine the reference interval for WBC counts in Holstein dairy cows from herds with high seroprevalence for anti-bovine leukemia virus (BLV) antibodies, analyze the correlation of total WBC counts and blood proviral load (bPVL) in BLV-infected animals, and determine whether total WBC count can be used a hematologic marker for in vivo infection. ANIMALS 307 lactating cows from 16 dairy herds with high BLV seroprevalence. PROCEDURES Blood samples were collected for assessment of plasma anti-BLV p24 antibody concentration (all cows), manual determination of WBC count (161 BLV-seronegative cows from 15 herds), and evaluation of bPVL (146 cows from another herd). RESULTS The WBC count reference interval (ie, mean ± 2 SD) for BLV-seronegative dairy cows was 2,153 to 11,493 cells/μL. Of the 146 cows used to analyze the correlation between WBC count and bPVL, 107 (73%) had WBC counts within the reference interval; of those cows, only 21 (19.6%) had high bPVL. Most cows with high WBC counts (35/39) had high bPVL. Mean WBC count for cows with high bPVL was significantly higher than values for cows with low or undetectable bPVL. White blood cell counts and bPVL were significantly (ρ = 0.71) correlated. CONCLUSIONS AND CLINICAL RELEVANCE These data have provided an updated reference interval for WBC counts in Holstein cows from herds with high BLV seroprevalence. In dairy cattle under natural conditions, WBC count was correlated with bPVL; thus, WBC count determination could be a potential tool for monitoring BLV infection levels in attempts to control transmission.

Epidemiological features of BLV natural infection

Dairy farms are heavily infected with Bovine Leukemia Virus (BLV) in Argentina and many other countries, where a control strategy should be design based on the behavior of natural. We conducted a series of studies with the aim to better understand the epidemiology of BLV. Infected new born calves were present in 3 studied farms (8.3%-11%). Proviral load (PVL) was very low in 9/10 new born analyzed calves but rapidly augmented during the first months of age. Cross-sectional studies showed that the rate of high PVL between seroreactors raised together with the prevalence, from 20.2% at 8 months of age to 44.4% in 26 months heifers, with similar levels to adult lactating cows of the same farm. Low fluctuation of blood PVL was observed on animals with naturally-acquired infections. We also observed 10 seroconversions between young heifers with an initial elevation of PVl. The presence of provirus in colostrum was significantly correlated with blood PVL (p≤0.0001). Provirus in milk was detectable in bulk tank (17.2%) and individual samples (40.4%). Colostrum of individual cows showed different provirus/antibodies dual profiles that permit to speculate about different infective/protective potential among infected animals. These findings suggest animals would be exposed to the infective challenge since a very young age. Consequently, it must be control as soon as possible after birth. The main focus should be put on the new-born infected calves that could play the role of main propagators together with the putative oral exposition through colostrum and milk.