Bovine Leukemia Virus molecular detection and associated factors among dairy herd workers in Antioquia, Colombia.

The Bovine Leukemia Virus (BLV) affects mainly cattle, is transmitted by exposure to contaminated biological fluids, and generates lymphomas in 5 % of infected animals. The zoonotic potential of BLV has been studied, and it is currently unknown if it circulates in human workers on dairy herds in Antioquia. Objective: To determine the frequency of BLV detection, the genotypes of the virus, and the factors associated with its detection in workers for dairy herds in Antioquia, Colombia. Through a cross-sectional study in 51 dairy herds, 164 adults were recruited. A peripheral blood sample was collected from each participant for molecular detection of the BLV env and tax genes, and associated factors were explored through bivariate and multivariate mixed Poisson model analyses. The analysis showed that 82 % (134/164) of the participants were men, with an average age of 40. Using qPCR, the constitutive gene GAPDH was amplified to evaluate the presence of amplification inhibitors in the DNA samples. Using nested PCR, the amplification of the env viral gene was obtained in 13 % (22/164) of the total samples analyzed, while all the samples tested negative for tax. The amplicons of the env gene were sequenced, and the identity compatible with BLV was verified by BLAST analysis (NCBI). Using molecular phylogeny analysis, based on maximum likelihood and haplotype network analysis, it was identified that BLV genotype 1 is present in the evaluated population. 16 % (26/164) of the participants reported having ever had an accident with surgical material during work with cattle; this variable was associated with BLV positivity even after adjusting for other variables (PRa =2.70, 95 % CI= 1.01- 7.21). Considering that other studies have reported the circulation of BLV genotype 1 in cattle from this same region and the present report in humans from dairy herds, the results suggest a possible zoonotic transmission of BLV genotype 1 in Antioquia, reinforcing the need to continue investigating to determine the potential role of this virus as an etiological agent of disease in livestock farmers in the department.

Characterization and application of recombinant Bovine Leukemia Virus Env protein.

The Bovine Leukemia Virus (BLV) Envelope (Env) glycoprotein complex is instrumental in viral infectivity and shapes the host's immune response. This study presents the production and characterization of a soluble furin-mutated BLV Env ectodomain (sBLV-EnvFm) expressed in a stable S2 insect cell line. We purified a 63 kDa soluble protein, corresponding to the monomeric sBLV-EnvFm, which predominantly presented oligomannose and paucimannose N-glycans, with a high content of core fucose structures. Our results demonstrate that our recombinant protein can be recognized from specific antibodies in BLV infected cattle, suggesting its potential as a powerful diagnostic tool. Moreover, the robust humoral immune response it elicited in mice shows its potential contribution to the development of subunit-based vaccines against BLV.

Seroprevalence of reproductive and infectious diseases in cattle: the case of Madre de Dios in the Peruvian southeastern tropics.

OBJECTIVE: The objective of this study was to determine the seroprevalence of reproductive and infectious diseases in tropical cattle in the Tambopata and Tahuamanu Provinces in the department of Madre de Dios, Peru. SAMPLE: 156 bovines from 7 cattle farms were sampled. These farms used exclusive grazing for food and natural mating for reproduction and did not have sanitary or vaccination programs. METHODS: The serum of blood samples was subjected to ELISA with commercial kits for the detection of antibodies against Neospora caninum, Mycobacterium avium subsp paratuberculosis (MAP), Leptospira interrogans, pestivirus bovine viral diarrhea virus-1, retrovirus bovine leukemia virus (BLV), orbivirus bluetongue virus (BTV), and herpesvirus bovine herpes virus-1 (BHV). The data were analyzed by means of association tests with χ2 (P < .05) and Spearman rank correlation (P < .05) in the SPSS v.15.0 software (IBM Corp). RESULTS: A low prevalence of antibodies to L interrogans, N caninum, M avium subsp paratuberculosis, bovine viral diarrhea virus-1 was found, but it was high to BTV, BLV, and BHV (100%, 53.85%, and 72.44%, respectively). The presence of BLV and BHV was higher in the Las Piedras District, bovines less than 5 years old, and cattle with breed characteristics of zebu and crossbred (P < .01). In addition, there was a significant correlation between both infections, showing 83.3% of BLV positivity that were also BHV positive (P < .01). CLINICAL RELEVANCE: The high prevalence of antibodies to BTV, BHV, and BLV could be due to livestock management practices, direct contact with infected animals, and variation of the presence of vectors and natural reservoirs in the context of climate change in the tropics.

Altered apoptosis and proliferation in milk cells and PBMc from BLV-infected bovines with different proviral loads: Possible role of the BCL-2 family proteins, TNF-alpha, and receptors.

Bovines infected by bovine leukemia virus (BLV) are characterized by presenting low proviral load (LPL) or high proviral load (HPL). It is reported that animals with HPL in peripheral blood mononuclear cells (PBMCs) present a decrease in apoptosis, an increase in viability and the proliferation rate, while animals that maintain an LPL have an intrinsic ability to control the infection, presenting an increased apoptosis rate of their PBMCs. However, there is little information on the effect of BLV on these mechanisms when the virus infects somatic milk cells (SC). This study investigates the mechanisms underlying apoptosis in milk and blood from BLV-infected animals with HPL and LPL. Relative levels of mRNA of tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNF-RI), TNF receptor 2 (TNF-RII), anti-apoptotic B-cell lymphoma 2 protein (Bcl-2), and pro-apoptotic Bcl-2-like protein 4 (Bax) were measured in SC and PBMCs using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. A significant decrease in the expression of TNF-α in SC from HPL animals vs non-infected bovines was observed, but the infection in SC with BLV did not show a modulation on the expression of TNF receptors. A significant increase in TNF-RI expression in PBMCs from HPL bovines compared to LPL bovines was observed. No significant differences in PBMCs between HPL and LPL compared to non-infected animals concerning TNF-α, TNF-RI, and TNF-RII expression were found. There was a significant increase of both Bcl-2 and Bax in SC from LPL compared to non-infected bovines, but the Bcl-2/Bax ratio showed an anti-apoptotic profile in LPL and HPL bovines compared to non-infected ones. Reduced mRNA expression levels of Bax were determined in the PBMCs from HPL compared to LPL subjects. In contrast, BLV-infected bovines did not differ significantly in the mRNA expression of Bax compared to non-infected bovines. Our data suggest that the increased mRNA expression of Bax corresponds to the late lactation state of bovine evaluated and the exacerbated increase of mRNA expression of Bcl-2 may be one of the mechanisms for the negative apoptosis regulation in the mammary gland induced by BLV infection. These results provide new insights into the mechanism of mammary cell death in HPL and LPL BLV-infected bovine mammary gland cells during lactation.

Seroprevalence and detection of bovine leukosis virus in semen from breeding bulls in Costa Rica.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a persistent infection and the most important neoplastic disease in cattle. It is spread primarily by transferring infected lymphocytes through blood from carriers to healthy animals. The present study is aimed at determining the seropositivity of BLV in breeding bulls from Costa Rica and at detecting for the first time in the country BLV DNA in bull semen. Between May 2011 and August 2018, 379 blood and 133 semen samples were collected from bulls distributed in 118 farms. The serum was analyzed by an enzymatic immunoassay and the semen by polymerase chain reaction and sequencing. BLV seropositivity was 43.5% (165/379), while 64.4% (76/118) of the farms had positive reactors. Holstein (75.7%) and Jersey (73.0%) breeds showed the highest seropositivity. In addition, Bos taurus bulls (68.1%), older than seven years (50.0%), and those belonging to dairy farms (75.5%) had higher seropositivity compared to Bos indicus (17.7%), younger than seven years (42.2%), and those from beef farms (15.5%), respectively. Moreover, Bos taurus bulls had a higher risk of being seropositive than Bos indicus (OR = 3.4; 95% CI: 1.7-6.8). BLV DNA was found in one semen sample (2.5%; 1/40) from a seropositive bull. The importance of serum and molecular BLV screening in semen samples and the potential role of some risk factors associated with the disease, such as the bull's age, genotype, and type of livestock productive system, is argued in the present report.

Bovine leukosis virus, bovine viral diarrhea, and bovine neosporosis seroprevalence in specialized dairy herds in Antioquia-Colombia.

Enzootic bovine leukosis (EBL) is a chronic infectious disease caused by the bovine leukosis virus (BLV), a Deltaretrovirus. Bovine viral diarrhea (BVD) is an infectious disease caused by a pestivirus. Bovine neosporosis is caused by the obligate intracellular parasite Neospora caninum (Nc). These pathogens can have horizontal (postnatal) or vertical (transplacental) transmissions and affect the productive and reproductive performance of infected bovines. This work aimed to detect BLV, BVD, and Nc seroprevalence in specialized dairy cattle from the north, east, and Aburrá Valley regions of the Department of Antioquia, the highest in milk production regions in Antioquia. A total of 599 blood samples, obtained from 53 specialized dairy cattle herds, were evaluated by the ELISA test. The results revealed a seroprevalence of 41.13% for BLV (242/599), 28.48% (163/599) for Nc, and 22.7% (132/599) for BVD. Regarding the regional seroprevalence evaluation, BLV was found in 47.02% of the samples from the east, 36.87% from the north, and 46.02% from the Aburrá Valley. Nc was found in 31.03% of the samples from the east, 24.26% from the north, and 36.63% from Aburrá Valley. BVD was found in 21.62% of the samples from the east, 25.03% from the north region, and 10.39% of the samples from the Aburrá Valley. It is highlighted by these results that the north region, with the highest milk production in Antioquia, had the lowest BLV and Nc seroprevalences but the highest seroprevalence of BVD. BLV has increased in Antioquia in recent years, and as an immunosuppressive infection, opportunities for other pathogens are created by it. A significant statistical difference was found in the average prevalence of the pathogens according to the municipality, cattle breed, and region of origin of the sample. The seroprevalence of these pathogens in specialized dairy herds in Antioquia can be classified as medium-low. However, it is recommended that biosecurity practices should be maximized to avoid the spread of these pathogens due to the variability detected in the region, municipality, breed group, and herd age. The rapid and efficient diagnosis of these three pathogens through reliable methodologies will allow for the control of dissemination in dairy herds.

Risk factors associated with enzootic bovine leukosis in Boyacá and Cundinamarca municipalities, Colombia.

Background: Enzootic bovine leukosis (EBL) is a lymphoproliferative disorder caused by the bovine leukemia virus (BLV), a virus of the Retroviridae family. The infection is distributed worldwide, and a high percentage of animals infected by the BLV are asymptomatic and act as carriers of the virus in many cattle populations. Aim: To identify the risk factors associated with EBL in the municipalities of Boyacá and Cundinamarca (Colombia). Methods: A simple descriptive cross-sectional study with random sampling was conducted. A total of 1,140 blood samples were taken from cattle (females and males) from the municipalities of Chiquinquirá, Ubaté, and San Miguel de Sema of different breeds and age groups. The samples were processed using the commercial ELISA SERELISA® BLV Ab Mono Blocking kit (sensitivity 97%, specificity 98%). The data were processed with the statistical programs WinEpi and Epi Info® version 7.2.4.0, estimating the prevalence ratio, implementing the chi-square test (p ≤ 0.05) and logistic regression. Results: A true prevalence (TP) and apparent prevalence (AP) of 23.61% and 22.7% in Ubaté, 19.22% and 18.1% in Chiquinquirá, and 15.61% and 14.3% in San Miguel de Sema, respectively, were established. Bovines 2-4 years old were the most prevalent in Ubaté and Chiquinquirá (37.5% and 21.21%, respectively), while in San Miguel de Sema individuals >4 years had the highest percentage of antibodies (18.3%). The Holstein breed had a higher prevalence in Ubaté and San Miguel de Sema (26.02% and 19.67%), and crossbreeds were more BLV-seroprevalence in Chiquinquirá (20.20%). In Ubaté, re-use of needles was identified as a risk factor, contaminated blood in needles is considered one of the main routes of transmission. On the other hand, manual milking was identified as a risk factor in San Miguel de Sema. Conclusion: The non-implementation of an individual needle per animal in Ubaté; the Holstein breed and manual milking in San Miguel de Sema were identified as risk factors for the presence of antibodies against the disease. EBL prevention and control plans should be established that focus on the implementation of management and sanitary practices based on herd biosecurity.

Seroprevalence and risk factors associated with bovine Leukemia virus infection in argentine beef cattle.

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.

Presence of co-infection between bovine leukemia virus and bovine herpesvirus 1 in herds vaccinated against bovine respiratory complex.

The aim of this study was molecular identification of bovine leukemia virus and possible co-infection with bovine respiratory disease complex (BRDC) viral agents in Mexican dairy herds. We collected 533 blood samples from cattle vaccinated against the BRDC virus in 9 states across Mexico. Peripheral blood leukocytes were removed and genetic material was extracted to detect bovine leukemia virus (BLV), bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), and bovine respiratory syncytial virus (BRSV) infection using polymerase chain reaction. We identified high BLV infection rates in 270 cattle (50.65%). One hundred and thirty-three cows (24.95%) tested positive for BoHV-1, of which 65 samples were positive for both viruses (BoHV-1 and BLV) and 68 were only positive for BoHV-1. Only 4 samples tested positive for BPIV-3 and no sample was positive for BVDV or BRSV. Relative risk and odds ratio analyses did not identify that the presence of BLV infection favors BoHV-1 co-infection in vaccinated herds.

Le but de cette étude était l'identification moléculaire du virus de la leucémie bovine et une éventuelle co-infection par des agents viraux du complexe des maladies respiratoires bovines (BRDC) dans des troupeaux laitiers mexicains. Nous avons recueilli 533 échantillons de sang de bovins vaccinés contre le virus BRDC dans neuf états du Mexique. Les leucocytes du sang périphérique ont été prélevés et le matériel génétique a été extrait pour détecter le virus de la leucémie bovine (BLV), le virus de l'herpès bovin 1 (BoHV-1), le virus de la diarrhée virale bovine (BVDV), le virus parainfluenza bovin 3 (BPIV-3), et le virus respiratoire syncytial bovin (BRSV) par réaction d'amplification en chaîne par la polymérase. Nous avons identifié des taux élevés d'infection par le BLV chez 270 bovins (50,65 %). Cent trente-trois bovins (24,95 %) ont été testés positifs pour le BoHV-1, desquels 65 échantillons étaient positifs pour les deux virus (BoHV-1 et BLV) et 68 étaient uniquement positifs pour le BoHV-1. Seuls quatre échantillons ont été testés positifs pour le BPIV-3 et aucun échantillon n'a été positif pour le BVDV ou le BRSV. Les analyses du risque relatif et des rapports de cotes n'ont pas identifié que la présence d'une infection par le BLV favorise la co-infection par le BoHV-1 dans les troupeaux vaccinés.(Traduit par les auteurs).

Bovine leukemia virus encoded blv-miR-b4-3p microRNA is associated with reduced expression of anti-oncogenic gene in vivo.

Bovine leukemia virus (BLV) is a retrovirus that causes malignant B-cell lymphoma in up to ten-percent of infected cattle. To date, the mechanisms of BLV linked to malignant transformation remain elusive. Although BLV-encoded miRNAs have been associated with the regulation of different genes involved in oncogenic pathways, this association has not been evaluated in cattle naturally infected with BLV. The objective of this study was to determine the relative expression of BLV-encoded miRNA blv-miR-b4-3p, the host analogous miRNA bo-miR-29a and a couple of potential target mRNAs (HBP-1 and PXDN, with anti-tumorigenic function in B-cells), in cattle naturally infected with BLV compared to uninfected animals (control group). We observed that PXDN was significantly downregulated in BLV-infected cattle (P = 0.03). Considering the similar expression of endogenous bo-miR-29a in both animal groups, the downregulation of PXDN in BLV-naturally infected cattle could be linked to blv-miR-b4-3p expression in these animals. Knowing that PXDN is involved in anti-tumoral pathways in B-cells, the results presented here suggest that blv-miR-b4-3p might be involved in BLV tumorigenesis during natural infection with BLV in cattle.

Descriptive cross-sectional study on major bovine diseases and associated risk factors in north-eastern Ecuadorian Amazon / Estudo transversal descritivo sobre as principais doenças bovinas e fatores de risco associados no nordeste da Amazônia equatoriana

Cattle raising is a crucial element of production systems in the tropics and subtropics. However, in recent years, global public health security has been threatened by disease emergence. In Orellana Province, livestock is the most important activity to generate economic income. Nevertheless, there is no available data about Animal Health status. With this objective, a study was performed to describe the major Bovine diseases recorded between 2011 to 2019, and the main Risk factors associated. Data on main Bovine diseases were retrieved from the World Animal Health Information System database. Whereas Bovine population data used to calculate the prevalence rates and confidence intervals were obtained from Ecuador's Ministry of Agriculture. By contrast, the Risk factors identified with an epidemiological questionnaire were applied to 300 livestock farmers. As a result, from 2011 to 2019 in Orellana has been confirmed: 90 cases of Infectious Bovine Rhinotracheitis (31.58%), Bovine Rabies by hematophagous bats (Desmodus rotundus), 83 cases (29.12%), Bovine viral diarrhea with 43 cases (15.10%), Brucellosis by Brucella abortus 35 cases, which was (12.28%), and 34 cases related to Enzootic bovine leukosis (11.92%). Overall, the prevalence rates ranged from (0.24 to 15.37%). In addition, farm size, presence of forest, herd, and paddock sizes, cutting frequency of forages, and other animal species were involved as Risk factors (OR = 3.15 to 11.75; 95% CI, 0.01 to 0.69). In conclusion, there are animal diseases with reproductive and neurologic symptomology and high-Risk factors implicated in the transmission. Consequently, space-temporal and seroprevalence epidemiological studies should be performed in Orellana.

A criação de gado é um elemento crucial dos sistemas de produção nos trópicos e subtrópicos. No entanto, nos últimos anos, a segurança da saúde pública global tem sido ameaçada pelo surgimento de doenças. Na província de Orellana, a pecuária é a atividade mais importante para gerar renda econômica. Contudo, não há dados disponíveis sobre o estado de saúde animal. Com este objetivo, foi realizado um estudo para descrever as principais doenças dos bovinos registradas entre 2011 e 2019, além dos principais fatores de risco associados. Os dados sobre as principais doenças bovinas foram recuperados do banco de dados do World Animal Health Information System. Os dados da população bovina usados para calcular as taxas de prevalência e os intervalos de confiança foram obtidos do Ministério da Agricultura do Equador. Por outro lado, os fatores de risco identificados com um questionário epidemiológico foram aplicados a 300 criadores de gado. Como resultado, de 2011 a 2019 em Orellana foram confirmados: 90 casos (31,58%) de rinotraqueíte infecciosa bovina, 83 casos (29,12%) de raiva bovina por morcegos hematófagos (Desmodus rotundus), 43 casos (15,10%) de diarreia viral bovina, 35 casos (12,28%) de brucelose por Brucella abortus e 34 casos relacionados à leucose enzoótica bovina (11,92%). No geral, as taxas de prevalência variaram de 0,24 a 15,37%. Além disso, tamanho da fazenda, presença de floresta, tamanho do rebanho e dos piquetes, frequência de corte de forragens e outras espécies animais estiveram envolvidos como fatores de risco (OR = 3,15-11,75; IC 95% 0,01-0,69). Em conclusão, existem doenças animais com sintomatologia reprodutiva e neurológica e fatores de alto risco implicados na transmissão. Portanto, estudos epidemiológicos espaço-temporais e de soroprevalência devem ser realizados em Orellana.

Enzootic bovine leukosis in a cow

Clinical History: A 5-year-old, Holstein-Friesian dairy cow was evaluated by a veterinary practitioner for a 30-day history of unilateral exophthalmos (Fig. 1). After 15 days, the cow presented lameness followed by progressive weight loss and pelvic limbs paresis, culminating in persistent sternal recumbency (Fig. 2). The superficial inguinal lymph nodes were enlarged. Due to the poor prognosis, the cow was euthanized and submitted to a postmortem examination. Gross Findings: The cow was in poor body condition with mild amounts of subcutaneous and visceral fat stores. The oral and conjunctival mucous membranes were pale. There was severe exophthalmos in the right eye, caused by a soft, homogenous, white to yellow mass (6 cm in diameter) (Fig. 3) in the retrobulbar space. Similar irregular masses were seen in the left renal pelvis, partially effacing the renal parenchyma, and in the epidural space, circumferentially surrounding the pachymeninges (extradural location) (Fig. 4) of the lumbar segment of the spinal cord. The superficial inguinal lymph nodes (supramammary) were markedly enlarged and, on the cut surface, had homogenous white to yellow discoloration and loss of the corticomedullary junction. Multifocal areas of the abomasum wall were moderately thickened and expanded by a soft, homogenous, white to yellow masses. No significant alterations were observed in other organs. Follow-up questions: Morphologic diagnosis? Etiological agent? Name of the condition? Probable pathogenesis pathways?

Seroprevalence and risk factors associated with leukosis in cattle from Villlavicencio, Colombia / Soroprevalência e fatores de risco associados à leucose em bovinos de Villlavicencio, Colômbia

Bovine leukosis is caused by an oncogenic virus of the genus Deltaretrovirus, causing losses associated with decreased production indicators and restrictions on exports of cattle and cattle products. The disease has a prolonged incubation period of between 1-5 years and the antibodies can be detected 2-3 weeks post infection. The disease can present asymptomatically, and develop persistent lymphocytosis or lymphosarcoma. The objective of this study was to estimate the prevalence and risk factors associated with bovine leukosis in Villavicencio, Colombia. Blood samples were taken from 636 animals, and obtained randomly from 24 herds. The samples were analysed using a Competition ELISA kit for the detection of anti-gp51 antibodies. Information on possible risk factors was collected, then OR and X2 were calculated, and statistically significant with p < 0.05 variables were included in a linear regression multivariate analysis. The general seroprevalence was 24.6% and the herd seroprevalence was 83.3%. The seroprevalence was 21.3% in males and 25.0% in females. The risk factors identified were abortion, non-bearing cows, artificial insemination, and use of common needles, Creole breed and participation in cattle exhibitions. The study confirmed the presence of bovine leukosis associated with reproductive and management factors.(AU)

A leucose bovina é causada por um vírus oncogênico do gênero Deltaretrovirus, causando prejuízos associados à queda dos indicadores produtivos e restrições à exportação de bovinos e derivados.Adoença tem um período de incubação prolongado entre 1 e 5 anos e os anticorpos podem ser detectados 2 a 3 semanas após a infecção. A doença pode se apresentar de forma assintomática, e evoluir para linfocitose persistente ou linfossarcoma. O objetivo do estudo foi estimar a prevalência e os fatores de risco associados à leucose bovina em Villavicencio, Colômbia. Amostras de sangue foram coletadas de 636 animais, obtidos aleatoriamente de 24 rebanhos.As amostras foram analisadas com o kit Competition ELISA para detecção de anticorpos anti-gp51. Foram coletadas informações sobre possíveis fatores de risco, se realizo um analise univariado entre as variáveis e a presença da seropositividad a leukosis bovina mediante o cálculo do OR e X2, as variáveis estatisticamente significativas com p<0,05 foram incluídas em uma análise multivariada de regressão linear. A soroprevalência geral foi de 24,6% e a soroprevalência do rebanho foi de 83,3%.Asoroprevalência foi de 21,3% em machos e 25,0% em fêmeas. Os fatores de risco identificados foram: aborto, vacas não reprodutivas, inseminação artificial e uso de agulha comum, raça crioula e exposições de gado. O estudo confirmou a presença de leucose bovina associada a fatores reprodutivos e de manejo.(AU)

Natural association between bovine leukemia virus and reproductive infectious diseases

Background: Enzootic bovine leukosis (EBL) is a widespread infectious disease caused by the bovine leukemia virus (BLV), which results in immune system dysfunction. The resulting immunosuppression may lead to an increased prevalence of other diseases. Dairy cows infected have altered immune function associated with decreased milk production and shortened lifespan and decreased immune response to immunization. BLV infection, however, is often asymptomatic, so any connection between subclinical infection and common reproductive diseases remains unknown. This study aimed to describe the relationship between naturally occurring subclinical BLV and infectious reproductive diseases seroconversion in the field. Materials, Methods & Results: The diseases investigated included Bovine viral diarrhoea (BVD), Bovine alfaherpesvirus 1 (BoHV-1), Bovine gammaherpesvirus 4 (BoHV-4), Chlamydiosis, Leptospirosis, Brucellosis and Neosporosis in dairy cattle. Six hundred fifty-five sera samples from the northern and south-central regions of Uruguay, from asymptomatic female Holstein and Holstein crosses without a history of vaccination against reproductive diseases were processed using reference diagnostic methods (Seronautalization, ELISA, MAT, Rose Bengal Plate test). The seroprevalence of BLV was 20.0%. Seroprevalence of reproductive diseases BVD, BoHV-1 and BoHV-4 were 99.3%, 41.2% and 27.3% of the populations, respectively, and the total seroprevalence of Leptospirosis, bovine Neospora caninum and Chlamydiosis were 19.8%, 29.8% and 33.0% respectively. The results revealed positive associations between naturally contracted BLV and the presence of antibodies against BoHV-1 (P = 0.002), as well as between naturally contracted BLV and presence of antibodies against Leptospira spp. (P = 0.028). Discussion: BLV infection can impact innate and adaptive immune system cells and alter the proper functioning of uninfected cells. BLV infection may also induce changes in the complex balance of cytokine expression, cell proliferation, and programmed cell death in T- and B-lymphocytes, which is critical for immune competence and effective response to infectious challenges. The progression of BLV infection has a substantial effect on host defense mechanisms. Indeed, lowmagnitude serologic responses to a commercial foot-and-mouth disease vaccine and a J5 Escherichia coli vaccine have been observed. These results are supported by recent trial studies showing a reduced immune response to vaccination against BoHV-1 and Leptospira spp. in asymptomatic animals infected with BLV. These are 2 of the most prevalent infectious reproductive diseases in cattle worldwide, and our results provide evidence that a link between BLV and susceptibility to these diseases may exist. Although there is evidence of the co-occurrence of these diseases, it remains unknown whether there is a direct or indirect effect of BLV on infertility, embryonic loss, or abortion. Another possibility is that natural infection with these reproductive pathogens (BoHV-1, Leptospira, or others) promotes BLV expression, negatively affecting the farms where these pathogens are endemic. Considering the high seroprevalence of BLV in dairy herds in North and South America where the infection is endemic, it was explored BLV's role as an immunosuppressant by quantifying its co-occurrence with diseases that affect reproductive performance in breeding herds. Future work should clarify the role of BLV and the co-occurring pathogens in causing infertility or abortions.

Relationship of persistent lymphocytosis, antibody titers, and proviral load with expression of interleukin-12, interferon-γ, interleukin-2, interleukin-4, interleukin-10, and transforming growth factor-ß in cows infected with bovine leukemia virus from a high-prevalence dairy complex.

Bovine leukemia virus (BLV) subclinical infection promotes persistent lymphocytosis (PL), which is related to susceptibility and progression to lymphoma. Moreover, lymphocyte counts directly correlate with BLV antibody titers and proviral load, and cell immune responses are considered atypical due to immune suppression. In order to determine the relationship of PL, antibody titers, and proviral load with interleukin (IL)-12, interferon (IFN)-γ, IL-2, IL-4, IL-10, and transforming growth factor (TGF)-ß expression in a 3-month interval, 58 cows were selected (30 BLV+ and 28 BLV-) from a high-prevalence dairy herd to complete 3 monthly blood samplings for the assessment of PL, BLV antibody titers, BLV proviral load, and IL-12, IFN-γ, IL-2, IL-4, IL-10, and TGF-ß expression. At sampling conclusion, the BLV-infected cows were grouped according to PL, BLV proviral load, and BLV antibody titers as follows: BLV+PL+ (n = 16) and BLV+PL- (n = 14); high proviral load (HPL) (n = 18) and low proviral load (LPL) (n = 13); high antibody titers (HAT) (n = 17) and low antibody titers (LAT) (n = 14). The BLV+PL+ cows showed significantly higher proviral load and antibody titers than the BLV+PL- group; however, the former suggested spread presumably unrelated to lymphoma outcome, because HPL was observed in PL- cows in the last sampling. Consistent with the data, a higher antibody response strongly indicated BLV susceptibility since it was linked to PL+ occurrence and a cytokine profile compatible with immune suppression. Furthermore, a reversion to lower antibody titers was observed in cows with HPL far ahead of time, most likely due to long-term immune suppression. In addition, high expression of IL-10 and TGF-ß was associated with reduced IL-12, IFN-γ, IL-2, and IL-4 expression alongside PL, HAT, and HPL in BLV-infected cows, suggesting an IL-10- and TGF-ß-induced immune suppression. The IL-10 expression was increasing throughout, implying disease progression, as described. In conclusion, the proliferative expansion of lymphocytes known as PL might enhance a regulatory-rich cell population (Bregs and/or Tregs) that secretes IL-10 and TGF-ß, leading to immune suppression. Further studies must be conducted regarding the types of regulatory cells involved in BLV-induced immune suppression.

L'infection subclinique par le virus de la leucémie bovine (BLV) favorise une lymphocytose persistante (PL), qui est liée à la susceptibilité et à la progression vers le lymphome. De plus, le nombre de lymphocytes est directement corrélé aux titres d'anticorps BLV et à la charge provirale, et les réponses immunitaires cellulaires sont considérées comme atypiques en raison de la suppression immunitaire. Afin de déterminer la relation entre PL, les titres d'anticorps et la charge provirale avec l'interleukine (IL)-12, l'interféron (IFN)-γ, l'IL-2, l'IL-4, l'IL-10 et l'expression du facteur de croissance transformant (TGF)-ß dans un intervalle de 3 mois, 58 vaches ont été sélectionnées (30 BLV+ et 28 BLV−) à partir d'un troupeau laitier à forte prévalence pour compléter trois prélèvements sanguins mensuels pour l'évaluation de PL, des titres d'anticorps BLV, de la charge provirale BLV et l'expression d'IL-12, IFN-γ, d'IL-2, d'IL-4, d'IL-10 et TGF-ß. À la fin de l'échantillonnage, les vaches infectées par le BLV ont été regroupées en fonction du PL, de la charge provirale du BLV et des titres d'anticorps du BLV comme suit : BLV+PL+ (n = 16) et BLV+PL− (n = 14); charge provirale élevée (HPL) (n = 18) et charge provirale faible (LPL) (n = 13); titres d'anticorps élevés (HAT) (n = 17) et titres d'anticorps faibles (LAT) (n = 14). Les vaches BLV+PL+ ont montré une charge provirale et des titres d'anticorps significativement plus élevés que le groupe BLV+PL−; cependant, le premier suggère une propagation vraisemblablement sans rapport avec l'issue du lymphome, car HPL a été observé chez les vaches PL− lors du dernier échantillonnage. Conformément aux données, une réponse anticorps plus élevée indiquait fortement une sensibilité au BLV puisqu'elle était liée à l'apparition de PL+ et à un profil de cytokines compatible avec la suppression immunitaire. De plus, un retour à des titres d'anticorps plus faibles a été observé chez les vaches atteintes de HPL bien avant le temps, probablement en raison d'une immunosuppression à long terme. De plus, une expression élevée d'IL-10 et de TGF-ß était associée à une expression réduite d'IL-12, d'IFN-γ, d'IL-2 et d'IL-4 aux côtés de PL, HAT et HPL chez les vaches infectées par le BLV, suggérant une immunosuppression induite par IL-10 et le TGF-ß. L'expression d'IL-10 augmentait tout au long, impliquant une progression de la maladie, comme décrit. En conclusion, l'expansion proliférative des lymphocytes connus sous le nom de PL pourrait renforcer une population de cellules riches en régulation (Bregs et/ou Tregs) qui sécrète d'IL-10 et du TGF-ß, conduisant à une suppression immunitaire. D'autres études doivent être menées sur les types de cellules régulatrices impliquées dans la suppression immunitaire induite par le BLV.(Traduit par Docteur Serge Messier).

A safe and effective vaccine against bovine leukemia virus.

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.

Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus.

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.

Diagnosis failure of bovine leukosis: serology variation during the peripartum period.

This study demonstrates the influence of pregnancy on serum diagnosis of enzootic bovine leukosis (EBL), emphasizing the importance of routine testing to maintain herd health. For this, 143 pregnant cows were sampled in duplicate (30 days before and 15 days after calving). For EBL diagnosis, samples were submitted to agar gel immunodiffusion testing (AGID). Different results were observed before and after delivery in seventy-six serum samples (53.15%), indicating variations in the levels of serum globulins in the blood during the peripartum period. Therefore, using a single sample for serological diagnosis during the birth season might not represent the correct infection status of animal health due to physiological variations in antibody concentrations.

Evidence of bovine leukemia virus circulating in sheep and buffaloes in Colombia: insights into multispecies infection.

Bovine leukemia virus (BLV) is the causative agent of leukemia/lymphoma in cattle. However, previous evidence has shown its presence in other species of livestock as well as in humans, suggesting that other species can be accidental hosts of the virus. In viral infections, receptors that are common to different animal species are proposed to be involved in cross-species infections. For BLV, AP3D1 has been proposed to be its receptor, and this protein is conserved in most mammalian species. In Colombia, BLV has been reported in cattle with high prevalence rates, but there has been no evidence of BLV infections in other animal species. In this study, we tested for the virus in sheep (n = 44) and buffaloes (n = 61) from different regions of Colombia by nested PCR, using peripheral blood samples collected from the animals. BLV was found in 25.7% of the animals tested (12 buffaloes and 15 sheep), and the results were confirmed by Sanger sequencing. In addition, to gain more information about the capacity of the virus to infect these species, the predicted interactions of AP3D1 of sheep and buffaloes with the BLV-gp51 protein were analyzed in silico. Conserved amino acids in the binding domains of the proteins were identified. The detection of BLV in sheep and buffaloes suggests circulation of the virus in multiple species, which could be involved in dissemination of the virus in mixed livestock production settings. Due to the presence of the virus in multiple species and the high prevalence rates observed, integrated prevention and control strategies in the livestock industry should be considered to decrease the spread of BLV.

Use of pooled serum samples to assess herd disease status using commercially available ELISAs.

Pooled samples are used in veterinary and human medicine as a cost-effective approach to monitor disease prevalence. Nonetheless, there is limited information on the effect of pooling on test performance, and research is required to determine the appropriate number of samples which can be pooled. Therefore, this study aimed to evaluate the use of pooled serum samples as a herd-level surveillance tool for infectious production-limiting diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL) and Neospora caninum (NC), by investigating the maximum number of samples one can pool to identify one positive animal, using commercial antibody-detection ELISAs. Four positive field standards (PFS), one for each disease, were prepared by pooling highly positive herd-level samples diagnosed using commercially available ELISA tests. These PFS were used to simulate 18 pooled samples ranging from undiluted PFS to a dilution representing 1 positive in 1,000 animals using phosphate-buffered saline as diluent. A 1:10 dilution of the PFS resulted in positive results for IBR, BVD and EBL. Moreover, for IBR and BVD, results were still positive at 1:100 and 1:30 dilutions, respectively. However, for NC, a lower dilution (8:10) was required for a seropositive result. This study indicates that, at herd-level, the use of pooled serum is a useful strategy for monitoring infectious diseases (BVD, IBR and EBL) but not NC, using readily available diagnostic assays.