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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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Previous attempts to develop a vaccine against bovine leukemia virus (BLV) have not been successful because of inadequate or short-lived stimulation of all immunity components. In this study, we designed an approach based on an attenuated BLV provirus by deleting genes dispensable for infectivity but required for efficient replication. The ability of the vaccine to protect from natural BLV infection was investigated in the context of dairy productive conditions in an endemic region. The attenuated vaccine was tested in a farm in which the prevalence rose from 16.7% in young cattle at the beginning of the study to more than 90% in adult individuals. Sterilizing immunity was obtained in 28 out of 29 vaccinated heifers over a period of 48 months, demonstrating the effectiveness of the vaccine. As indicated by the antiviral antibody titers, the humoral response was slightly reduced compared to wild-type infection. After initial post-vaccination bursts, the proviral loads of the attenuated vaccine remained most frequently undetectable. During the first dairy cycle, proviral DNA was not detected by nested-PCR in milk samples from vaccinated cows. During the second dairy cycle, provirus was sporadically detected in milk of two vaccinated cows. Forty-two calves born from vaccinated cows were negative for proviral DNA but had antiviral antibodies in their peripheral blood. The attenuated strain was not transmitted to sentinels, further supporting the safety of the vaccine. Altogether, these data thus demonstrate that the vaccine against BLV is safe and effective in herd conditions characterized by a very high incidence. This cost-effective approach will thus decrease the prevalence of BLV without modification of production practices. After facing a series of challenges pertaining to effectiveness and biosafety, the vaccine is now available for further large-scale delivery. The different challenges and hurdles that were bypassed may be informative for the development of a vaccine against HTLV-1.

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Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants-a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.

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Fetal bovine serum (FBS) used in cell culture may be contaminated with adventitious agents, which can affect the production of biologicals and the results of clinical laboratory tests. We carried out a retrospective study to determine the incidence of adventitious agent contamination of Argentinean irradiated FBS dating from 2015 to 2019. We analyzed FBS batches for mycoplasma and adventitious viruses (bovine pestiviruses, bovine adenovirus, bluetongue virus, bovine parainfluenza virus 3, rabies virus, bovine parvovirus, bovine herpesvirus 1, bovine respiratory syncytial virus, and reovirus). Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. The presence of neutralizing antibodies against pestiviruses was determined. Molecular analyses indicated that frequencies of mycoplasma and pestiviruses in FBS were 14% and 84%, respectively. All of the batches were seronegative for pestiviral antibodies. After cell passages, all FBS samples were negative for hemadsorbent agents and by immunofluorescence for all of the viral species analyzed; PCR assays were negative for mycoplasma and pestiviruses. Our results demonstrate that, of all adventitious agents tested, local FBS batches only had traces of mycoplasma and pestiviruses; gamma irradiation was effective in inactivating them.

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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) infection is omnipresent in dairy herds causing direct economic losses due to trade restrictions and lymphosarcoma-related deaths. Milk production drops and increase in the culling rate are also relevant and usually neglected. The BLV provirus persists throughout a lifetime and an inter-individual variation is observed in the level of infection (LI) in vivo. High LI is strongly correlated with disease progression and BLV transmission among herd mates. In a context of high prevalence, classical control strategies are economically prohibitive. Alternatively, host genomics studies aiming to dissect loci associated with LI are potentially useful tools for genetic selection programs tending to abrogate the viral spreading. The LI was measured through the proviral load (PVL) set-point and white blood cells (WBC) counts. The goals of this work were to gain insight into the contribution of SNPs (bovine 50KSNP panel) on LI variability and to identify genomics regions underlying this trait. RESULTS: We quantified anti-p24 response and total leukocytes count in peripheral blood from 1800 cows and used these to select 800 individuals with extreme phenotypes in WBCs and PVL. Two case-control genomic association studies using linear mixed models (LMMs) considering population stratification were performed. The proportion of the variance captured by all QC-passed SNPs represented 0.63 (SE ± 0.14) of the phenotypic variance for PVL and 0.56 (SE ± 0.15) for WBCs. Overall, significant associations (Bonferroni's corrected -log10p > 5.94) were shared for both phenotypes by 24 SNPs within the Bovine MHC. Founder haplotypes were used to measure the linkage disequilibrium (LD) extent (r2 = 0.22 ± 0.27 at inter-SNP distance of 25-50 kb). The SNPs and LD blocks indicated genes potentially associated with LI in infected cows: i.e. relevant immune response related genes (DQA1, DRB3, BOLA-A, LTA, LTB, TNF, IER3, GRP111, CRISP1), several genes involved in cell cytoskeletal reorganization (CD2AP, PKHD1, FLOT1, TUBB5) and modelling of the extracellular matrix (TRAM2, TNXB). Host transcription factors (TFs) were also highlighted (TFAP2D; ABT1, GCM1, PRRC2A). CONCLUSIONS: Data obtained represent a step forward to understand the biology of BLV-bovine interaction, and provide genetic information potentially applicable to selective breeding programs.

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In this work, we studied seven groups of pregnant heifers from a consortium of dairy farms heavily infected with bovine leukemia virus (BLV). ELISA testing showed that the seroprevalence ranges of BLV in heifers between 36.1 and 66.5 %. No significant differences in proviral load were found when comparing heifers with adult cattle. Before their first delivery, more than 9.8 % of heifers show a high proviral load. Because BLV infection can occur during the first two years of life, the rationale of any strategy should be to take action as early as possible after birth.

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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 dam's blood and colostrum from two heavily infected dairy farms, and investigated the correlation between the dam's 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.

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The most used and reliable indicator of Equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV infected animals, allowing to identify a higher number of positive horse cases. Thus, ELISA assays can be useful tools in EIA control and eradication.

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The most used and reliable indicator of equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV infected animals, allowing to identify a higher number of positive horse cases. Thus, ELISA assays can be useful tools in EIA control and eradication.

El mejor indicador de la infección por el virus de la anemia infecciosa equina (Equine infectious anemia virus, EIAV) es la detección de anticuerpos específicos en el suero del caballo. En el presente trabajo se evaluó la capacidad de detección de anticuerpos contra EIAV de dos equipos de ELISA comerciales utilizando 302 muestras de suero equino, así como las ventajas potenciales de su uso como herramientas de screening. Ambos ensayos de ELISA presentaron 100 % de sensibilidad diagnóstica y una especificidad diagnóstica del orden de 92,3 a 94,3 %. Las muestras discordantes fueron analizadas por inmunoblot. Los resultados mostraron que las dos pruebas ELISA son muy eficientes para detectar animales infectados por EIAV, al permitir identificar un mayor número de animales positivos que la prueba de inmunodifusión en gel de agar, oficialmente aprobada en la República Argentina para la certificación de los animales. Las pruebas de ELISA constituyen herramientas muy útiles en los programas de control y de erradicación de la infección por EIAV.

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The most used and reliable indicator of equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV infected animals, allowing to identify a higher number of positive horse cases. Thus, ELISA assays can be useful tools in EIA control and eradication.(AU)

El mejor indicador de la infección por el virus de la anemia infecciosa equina (Equine infectious anemia virus, EIAV) es la detección de anticuerpos específicos en el suero del caballo. En el presente trabajo se evaluó la capacidad de detección de anticuerpos contra EIAV de dos equipos de ELISA comerciales utilizando 302 muestras de suero equino, así como las ventajas potenciales de su uso como herramientas de screening. Ambos ensayos de ELISA presentaron 100 % de sensibilidad diagnóstica y una especificidad diagnóstica del orden de 92,3 a 94,3 %. Las muestras discordantes fueron analizadas por inmunoblot. Los resultados mostraron que las dos pruebas ELISA son muy eficientes para detectar animales infectados por EIAV, al permitir identificar un mayor número de animales positivos que la prueba de inmunodifusión en gel de agar, oficialmente aprobada en la República Argentina para la certificación de los animales. Las pruebas de ELISA constituyen herramientas muy útiles en los programas de control y de erradicación de la infección por EIAV.(AU)

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The most used and reliable indicator of Equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100

diagnostic sensitivity, and 92.3-94.3

diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV infected animals, allowing to identify a higher number of positive horse cases. Thus, ELISA assays can be useful tools in EIA control and eradication.

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Bovine leukemia virus (BLV) and human T-lymphotropic virus type 1 (HTLV-1) are closely related d-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 d-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.

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BACKGROUND: Bovine leukemia virus (BLV) is highly endemic in many countries, including Argentina. As prevention of the spread from infected animals is of primary importance in breaking the cycle of BLV transmission, it is important to know the pathophysiology of BLV infection in young animals, as they are the main source of animal movement. In this work, we determined the proviral load and antibody titers of infected newborn calves from birth to first parturition (36 months). RESULTS: All calves under study were born to infected dams with high proviral load (PVL) in blood and high antibody titers and detectable provirus in the colostrum. The PVL for five out of seven calves was low at birth. All animals reached PVLs of more than 1% infected peripheral blood mononuclear cells (PBMCs), three at 3 months, one at 6 months, and one at 12 months. High PVLs persisted until the end of the study, and, in two animals, exceeded one BLV copy per cell. Two other calves maintained a high PVL from birth until the end of the study. Antibody titers were 32 or higher in the first sample from six out of seven calves. These decayed at 3-6 months to 16 or lower, and then increased again after this point. CONCLUSIONS: Calves infected during the first week of life could play an active role in early propagation of BLV to susceptible animals, since their PVL raised up during the first 12 months and persist as high for years. Early elimination could help to prevent transmission to young susceptible animals and to their own offspring. To our knowledge, this is the first study of the kinetics of BLV proviral load and antibody titers in newborn infected calves.

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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.

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