Bovine leukemia virus (BLV) is a deltaretrovirus from the Orthoretrovirinae subfamily and Retroviridae family. BLV causes a persistent infection in cattle and in most cases this infection is asymptomatic [1]. In one third of infected animals the infection progresses to a state of persistent lymphocytosis (PL) and 1 to 10% of infected cattle develop lymphosarcoma [1]. BLV is distributed worldwide except for Western Europe, Oceania and few other countries (e.g., Israel), where BLV has been eradicated. The high prevalence of this agent is associated with a significant economic impact on dairy industry due to trade restrictions and replacement cost. Additionally, BLV negatively affects production (i.e., reduced milk production) by interfering with cattle immunity and increasing the risk of secondary infection and disease (i.e., pneumonia, diarrhoea, mastitis, etc.), as reviewed by Training et al., Bartlett et al. and Frie et al. [2,3,4].

During primary BLV infection, viral spread occurs via the production of virions able to infect new target cells (replicative infection stage) [5]. The expression of viral antigens induces a strong humoral and cytotoxic immune response, and such response is effective in limiting the virus replication cycle. Since anti-viral immune response persist throughout the animal’s life, during the chronic stage of infection, the integrated proviruses expand by mitosis of infected host cells carrying BLV provirus, by a process known as clonal expansion (mitotic infection stage) [5]. Supporting this hypothesis several studies support a lack of viral expression in cells with an integrated provirus and BLV transcription is almost undetectable in vivo, during the chronic stage of infection; however, the incubation of heparinized blood at 37 °C is sufficient to induce ex vivo virus reactivation in PL infected subjects [6]. Alternatively, it has been proposed that low levels of viral expression persist in a subpopulation of infected cells, and these cells are efficiently killed by the immune system [7]. In this regard, the balance between a virus attempting to proliferate and a strong immune pressure may drive the course of BLV infection in its natural host. In this scenario, the host immune response may be unable to destroy cells in which viral transcription has been completely silenced, and for that reason the viral reservoir cannot be cleared.

Here we present a case report describing the spontaneous reactivation of BLV infection in naturally infected cattle. Moreover, we performed a pilot study to investigate if a stressor stimuli, in this case the administration of a synthetic GC [dexamethasone (DEX)], had an effect inducing the reactivation of BLV chronic infection in cattle. We hypothesized that by interacting with BLV LTR and/or modulating host immunity, DEX may provoke BLV reactivation in its natural host.

Case presentation

Spontaneous replication of BLV in chronically infected cattle. In order to investigate if virus reactivation occurred during the course of chronic infection with BLV we followed-up for 328 days, one single Holstein cow (> 3 years; ID# 184) which was chronically infected with BLV and presented high-proviral loads. Blood samples from this animal were obtained at days 0, 20, 52, 82, 110, 160, 165, 167, 174, 181, 188, 216, 244 and 328, and the detection of viral RNA in plasma was used to assess virus reactivation in this animal under natural conditions. Refer to the Supplementary Materials for a detailed description of all materials and methods.

High-proviral load in animal 184 remained stable during the whole study [Mean number of BLV copies per μg of total DNA = 182,500 (154,240 - 210,767)] (Fig. 1). Interestingly, we observed three peaks of BLV pol RNA expression in animal 184 (at days 52, 160 and 188), suggesting different times of virus reactivation (Fig. 1). Although virus reactivation in this animal did not have considerable impact on the proviral loads, it might have impacted the levels of total lymphocytes and BLV-specific antibodies in blood and plasma, respectively (Fig. 1).

Fig. 1 Spontaneous virus reactivation during chronic BLV infection in cattle. The BLV proviral load (black), BLV pol RNA expression (red), level of BLV-sp antibodies (blue dotted line) and the absolute counts of lymphocytes (blue solid line) were followed-up in a single Holstein cow chronically infected with BLV. BLV DNA and RNA levels are expressed as Log 10 of copies per μg of total DNA and per ml of plasma, respectively (left axis). BLV-sp Abs were measured by ELISA and are expressed as percent of reactivity (right axis). The dotted horizontal black line represent the limit of detection of BLV qPCR (100 copies per μg). The Figure Insets correspond to a pilot study to assess the effect of a stressor stimuli on BLV infection in cattle. The BLV proviral load (Upper inset) and BLV pol RNA expression (Lower inset) were followed-up in four cattle infected with BLV; three of these cattle received DEX treatment (IDs# 177, 190 and 230) and one was used as an untreated control (Animal 184). All these parameters were measured in peripheral blood. BLV DNA and RNA levels are expressed as Log 10 of copies per μg of total DNA and per ml of plasma. The dotted horizontal black line represent the limit of detection of BLV qPCR (100 copies per μg). Black arrows represent DEX administrations Full size image

Two years after the end of this particular study, animal 184 was euthanized. At this time, we obtained blood from this animal and a limited amount of material from lymph-node, spleen, liver, kidney and bone marrow samples (see supplementary material for details). Although this animal presented very high-proviral load for several years (> 3 years) it did not present any clinical sign. Moreover, we did not detect BLV RNA expression in plasma beyond day 188 (Fig. 1 and Table 1). Interestingly, at necropsy, animal 184 presented detectable levels of BLV RNA in spleen; in contrast, BLV proviral DNA was detected in several tissues (i.e. spleen, lymph-node, liver, kidney and bone marrow) (Table 1).

Table 1 Detection of BLV pol gene (DNA) and transcript (RNA) in tissues from animal 184 Full size table

Dexamethasone treatment had no impact on virus expression during BLV chronic infection in cattle. Based on our observation regarding high and spontaneous expression of BLV RNA in animal 184, we performed a pilot study to investigate if a stressor stimuli, in this case the administration of a synthetic GC (DEX), had an effect inducing the reactivation of BLV chronic infection in cattle. We hypothesized that by interacting with BLV LTR and/or modulating host immunity, DEX may provoke BLV reactivation in its natural host. For this purpose we administered 0.1 mg/kg of DEX (IV) during five consecutive days to three Holstein cattle (> 3 years of age; IDs# 177, 190 and 230) chronically infected with BLV; of note, none of these animals had detectable levels of BLV RNA in plasma before the initiation of the study (at day − 50; lower inset from Fig. 1 and Additional file 1: Table S1). Animal 184 (our Case Report animal), did not receive DEX treatment and was used as a single control. All animals were followed-up for 28 days. Unexpectedly, at day 0 (before the first administration of DEX), we detected presence of BLV pol RNA in the plasma from all four animals, including animal 184 (control) (Lower inset of Fig. 1). Following DEX treatment, we observed similar kinetics of BLV RNA and DNA levels in animals 184 (control), 190 and 230. Viral RNA copy numbers in these three animals reached undetectable levels by day 7 (Lower inset of Fig. 1) and by day 14 BLV RNA levels in these animals rebounded and continued this trend by day 28. Despite these fluctuations in virus RNA expression, the proviral loads in these animals remained high and stable during the 28 days of study (Upper inset of Fig. 1). The fourth animal in this group (ID: 177) showed a slightly different pattern; BLV RNA levels remained stable during the first week following DEX treatment and then reached undetectable levels by day 14 after DEX treatment (Lower inset of Fig. 1). Besides being the animal with the lowest proviral load within the group, this animal (ID: 177) also showed the lowest levels of total lymphocytes.