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Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, an endemic disease in dairy cattle of Argentina. However, little is known about the seroprevalence of BLV in beef cattle. In this study, we conducted a cross-sectional study including farms from thirteen provinces of Argentina. A total of 5827 bovine serum samples were collected from 76 farms and analyzed using an in-house developed enzyme-linked immunosorbent assay. Information about herd management was collected through a questionnaire, and univariate and multivariate analyses were performed to detect risk factors associated with BLV infection. Herd-level seroprevalence was 71.05%, while the mean animal-level seroprevalence was 7.23% (median = 2.69%; min = 0, max = 75). Only two provinces had no positive BLV samples. The other eleven provinces showed more than 50% of their farms infected with BLV. The multivariate model revealed that BLV prevalence was significantly associated with the use of animals raised in the same farm for cattle replacement (P = 0.005), breeding cows by natural mating with a bull (P < 0.001), and weaning calves after 6 months of age (P = 0.011). This extensive study revealed that BLV seroprevalence in Argentine beef farms has increased during the last years and allowed identifying some management practices associated with BLV prevalence. These data deserve special attention because BLV infection in beef cattle seems to lead to a dissemination pattern similar to that observed during the last decades in dairy cattle, especially considering that Argentina is the sixth beef producer in the world, with about 5% of global beef production.

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Abstract Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Resumen El virus de la leucosis bovina (bovine leukemia virus [BLV]) es un importante agente patógeno del ganado que causa importantes pérdidas económicas en todo el mundo, especialmente en los rodeos lecheros. El uso de modelos animales proporciona información valiosa sobre la patogénesis de las infecciones virales. Se realizaron infecciones experimentales en ovejas usando sangre de bovinos infectados con BLV, clones moleculares de BLV infecciosos o células derivadas de tumores. La línea celular Fetal Lamb Kidney, persistentemente infectada con el BLV (FLK-BLV), es uno de los cultivos a largo plazo más utilizados para la producción permanente de virus. Las células FLK-BLV o las partículas virales obtenidas del sobrenadante del cultivo libre de células podrían usarse como fuente de provirus o de virus para infectar experimentalmente ovejas. En este trabajo, nuestro objetivo fue determinar la cantidad mínima de células FLK-BLV o de sobrenadante libre de células que contiene BLV necesaria para producir infección en ovejas. También evaluamos la cantidad de anticuerpos bovinos anti-BLV necesaria para neutralizar la infección. Observamos que las dos ovejas inoculadas experimentalmente con 5000 células FLK-BLV se infectaron, y que una de las dos ovejas que recibieron 500 células FLK-BLV se infectó. Ninguno de los animales inoculados con 50 células FLK-BLV mostró evidencia de infección. El sobrenadante FLK-BLV libre de células demostró ser infectivo en ovejas hasta la dilución 1:1000. Los anticuerpos BLV específicos mostraron actividad neutralizante, ya que ninguna de las ovejas se infectó. Por el contrario, los animales que recibieron un suero BLV negativo mostraron signos de infección por BLV. Estos resultados contribuyen a la optimización de un bioensayo en ovejas útil para caracterizar la infección por BLV.

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The authors wish to make the following corrections to this paper [...].

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Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

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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 for the first time 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.

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Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

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Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). Although efficient eradication programs have been successfully implemented in most European countries and Oceania, BLV infection rates are still high worldwide. BLV naturally infects cattle, inducing a persistent infection with diverse clinical outcomes. The virus infects lymphocytes and integrates a DNA intermediate as a provirus into the genome of the cells. Therefore, exposure to biological fluids contaminated with infected lymphocytes potentially spreads the virus. Vertical transmission may occur in utero or during delivery, and about 10% of calves born to BLV-infected dams are already infected at birth. Most frequently, transmission from dams to their offspring occurs through the ingestion of infected colostrum or milk. Therefore, although EBL is not a disease specific to the neonatal period, during this period the calves are at special risk of becoming infected, especially in dairy farms, where they ingest colostrum and/or raw milk either naturally or artificially. Calves infected during the first week of life could play an active role in early propagation of BLV to susceptible animals. This review discusses the main factors that contribute to neonatal BLV infection in dairy herds, as well as different approaches and management practices that could be implemented to reduce the risk of BLV transmission during this period, aiming to decrease BLV infection in dairy herds.

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BACKGROUND: Bovine 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. RESULTS: The 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 (Z(reactivity): 3.55, P < 0.001; Z(titer): 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%. CONCLUSIONS: We 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.

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