RESUMO
Bovine viral diarrhea virus (BVDV) infection has a significant economic impact on beef and dairy industries worldwide. Fetal infection with a non-cytopathic strain may lead to the birth of persistently infected (PI) offspring, which is the main event in the epidemiological chain of BVDV infection. This report describes the birth of 99 BVDV-PI heifer calves within 52 days of birth in a regular BVDV-vaccinated Brazilian dairy cattle herd and the subgenotypes of the infecting field strains. This study was conducted in a high-yielding open dairy cattle herd that frequently acquired heifers from neighboring areas for replacement. The farm monitors the birth of PI calves by screening all calves born using an ELISA (IDEXX) for BVDV antigen detection. All calves aged 1-7 days were evaluated. For positive and suspected results, the ELISA was repeated when the calves were close to one month old. A total of 294 heifer calves were evaluated between February and March 2021. Of these, 99 (33.7 %) had positive ELISA results and were considered PI calves. To evaluate the predominant BVDV species and subgenotypes in this outbreak, whole blood samples were collected from 31 calves born during the study period. All samples were submitted to the RT-PCR assay for the partial amplification of the BVDV 5'-UTR region, and these amplicons were subjected to nucleotide sequencing. Phylogenetic analysis identified BVDV-1b and BVDV-1d in 16 and 13 heifer calves, respectively. In two calves, it was not possible to determine the BVDV-1 subgenotype. Detection of PI animals and monitoring of circulating BVDV subgenotype strains are central to disease control. This study shows that regular BVDV vaccination alone may be insufficient to prevent BVDV infection in high-yielding open dairy cattle herds. Other biosecurity measures must be adopted to avoid the purchase of cattle with acute infections by BVDV or BVDV-PI, which can cause a break in the health profile of the herd and economic losses.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Vírus da Diarreia Viral Bovina Tipo 1 , Vírus da Diarreia Viral Bovina , Surtos de Doenças , Filogenia , Animais , Bovinos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Surtos de Doenças/veterinária , Feminino , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Brasil/epidemiologia , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/classificação , Vírus da Diarreia Viral Bovina/isolamento & purificação , Vírus da Diarreia Viral Bovina/imunologia , Genótipo , Vacinas Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Indústria de Laticínios , Vacinação/veterinária , Anticorpos Antivirais/sangueRESUMO
BACKGROUND: The knowledge on bovine vaccines against respiratory viruses on bronchoalveolar fluid cells is scarce. OBJECTIVE: To compare the effects of a commercial intranasal (IN) and intramuscular (IM) vaccine against bovine respiratory disease (BRD) complex viruses on bronchoalveolar fluid cells of healthy heifers. METHODS: 21 healthy heifers were assigned to three treatment groups: control (CO, N = 7), intranasally vaccinated (IN) (n = 7), and intramuscularly vaccinated (IM) (n = 7). The IN group received 1 mL of the commercial vaccine in each nostril once containing attenuated BoHV-1, bPIV-3, and BRSV. The IM group was vaccinated with two doses of 2 mL with an interval of 21 days of the commercial vaccine containing attenuated BoHV-1, bPIV-3, and BRSV plus inactivated BVDV. At day 0 (D0), before the first vaccine dose, and at D3, D7, and D21, after the last vaccine dose, airway bronchoscopy was performed to observe local irritation and collect bronchoalveolar lavage fluid (BALF). The bronchoalveolar count, cytological evaluation, bronchoalveolar cell oxidative metabolism, and total bronchoalveolar IgA and IgG were measured. RESULTS: The IN vaccine increased neutrophil cellularity at D7 and D21 and total IgA at D3 in BALF. Total IgA in BALF also increased at D3 and oxidative metabolism of bronchoalveolar cells at D21 lowered compared to the CO group. Following IM vaccination there was no alteration of immunoglobulins or cell oxidative metabolism in BALF. Both vaccines reduced the number of alveolar macrophages. CONCLUSION: Both vaccines induced bronchoalveolar inflammation during the establishment of the vaccine immunity, which was more expressive in the IN protocol.
Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Doenças dos Bovinos/prevenção & controle , Vacinação/veterinária , Administração Intranasal/efeitos adversos , Administração Intranasal/veterinária , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Líquido da Lavagem Broncoalveolar/química , Bovinos , Doenças dos Bovinos/virologia , Vírus da Diarreia Viral Bovina , Feminino , Infecções por Herpesviridae/prevenção & controle , Herpesvirus Bovino 1 , Imunoglobulina A , Imunoglobulina G , Injeções Intramusculares/efeitos adversos , Injeções Intramusculares/veterinária , Vírus da Parainfluenza 3 Bovina , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino , Infecções por Respirovirus/prevenção & controle , Infecções por Respirovirus/veterinária , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Virais/administração & dosagemRESUMO
Bovine viral diarrhoea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduced productive performance, growth retardation, reduced milk production and increased susceptibility to other diseases leading to early culling of animals. There are two primary methods used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programmes, but there are safety risks or insufficient protection, respectively, with these vaccines. Here, we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is the fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared with a conventional vaccine in cattle.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Doenças dos Bovinos , Vírus da Diarreia Viral Bovina , Vacinas Virais , Animais , Anticorpos Antivirais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Cobaias , Vacinas de Subunidades AntigênicasRESUMO
Interferon lambda (IFN-λ) plays an important role in inducing an antiviral state in mucosal surfaces and has been used as an effective biotherapeutic against several viral diseases. Here we performed a proof of concept study on the activity of a biologically active recombinant bovine IFN-λ (rIFN-λ) produced in eukaryotic cells against Bovine Viral Diarrhea Virus (BVDV) in cattle. We first confirmed the lack of toxicity of different concentrations of rIFN-λ in bovine peripheral blood cells and the safety of its subcutaneous application in calves in doses up to 12 IU/kg. The antiviral activity of the rIFN-λ against BVDV was assessed in calves that were inoculated with 6 IU/kg of rIFN-λ (n = 4) or mock-treated (n = 2) two days before and after challenge with a BVDV type-2 non-cytopathic strain. Mock-treated animals developed respiratory disease, shedded the virus from 4 to 7 days post-infection (dpi) and had viremia between 4 and 14 dpi. Conversely, calves treated with rIFN-λ did not develop clinical symptoms. The virus was not found in nasal secretions or sera. Only one animal had a positive viral RNA detection in serum at 7 dpi. All infected animals treated with rIFN-λ increased systemic type-I IFNs levels at 4 dpi. The antiviral treatment induced an earlier onset of the anti-BVDV neutralizing antibodies. Altogether, these results constitute the proof-of-principle of bovine IFN-λ as an antiviral biotherapeutic to protect cattle against the clinical disease caused by BVDV.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Doenças dos Bovinos/prevenção & controle , Vírus da Diarreia Viral Bovina/imunologia , Diarreia/veterinária , Imunização Passiva , Interferons/administração & dosagem , Fatores Etários , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Diarreia/prevenção & controle , Diarreia/virologia , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 2/imunologia , Feminino , Imunização Passiva/veterinária , Interferons/classificação , Interferons/genética , Interferons/imunologia , Estudo de Prova de Conceito , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Eliminação de Partículas ViraisRESUMO
BACKGROUND: Bovine Viral Diarrhea Virus (BVDV) is associated with gastrointestinal, respiratory and reproductive diseases of livestock across the world that causes continuous economic losses in the cattle industry. This virus can establish a persistent infection (PI) in calves after the fetal infection, making BVDV positive catle carriers and primary reservoirs which will constantly transmit the virus to healthy and new-born animals. For this reason, the detection of the PI animals in herds is the first line of prevention of the viral infection. RESULTS: In this study, PI animals were detected in five different regions of Colombia through RT-PCR techniques and confirmed by sequencing. BVDV genotypes were determined using one fragment of the 5'UTR. It was found a 7% BVDV prevalence in animals and 22% in farms; and genotype 1 was identified as a single genotype for all of the samples. All samples were BVDV 1a. CONCLUSION: This is the first report in Colombia with higher prevalence rates compared with other places in the world, turned out to be of great importance for the ranchers, the vaccine producers and animal health control parties.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Colômbia/epidemiologia , Vírus da Diarreia Viral Bovina/classificação , Feminino , Masculino , Filogenia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Saponin-based adjuvants are promising adjuvants that enhance both humoral and T-cell-mediated immunity. One of the most used natural products as vaccine adjuvants are Quillaja saponaria bark saponins and its fraction named Quil A®. Despite that, its use has been restricted for human use due to safety issues. As an alternative, our group has been studying the congener species Quillaja brasiliensis saponins and its performance as vaccine adjuvants, which have shown to trigger humoral and cellular immune responses comparable to Quil A® but with milder side effects. Here, we studied a semi purified aqueous extract (AE) and a previously little characterized saponin-enriched fraction (QB-80) from Q. brasiliensis as vaccine adjuvants and an inactivated virus (bovine viral diarrhea virus, BVDV) antigen co-formulated in experimental vaccines in mice model. For the first time, we show the spectra pattern of the Q. brasiliensis saponins by MALDI-TOF, a novel and cost-effective method that could be used to characterize different batches during saponins production. Both AE and QB-80 exhibited noteworthy chemical similarities to Quil A®. In addition, the haemolytic activity and toxicity were assessed, showing that both AE and QB-80 were less toxic than Quil A®. When subcutaneously inoculated in mice, both fractions promoted long-term strong antibody responses encompassing specific IgG1 and IgG2a, enhanced the avidity of IgG antibodies, induced a robust DTH reaction and significantly increased IFN-É£ production in T CD4+ and T CD8+ cells. Furthermore, we have proven herein that AE has the potential to promote dose-sparing, substantially reducing the dose of antigen required for the BVDV vaccines and still eliciting a mixed Th1/Th2 strong immune response. Based on these results, and considering that AE is a raw extract, easier and cheaper to produce than commercially available saponins, this product can be considered as candidate to be escalated from experimental to industrial uses.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Imunidade Celular/imunologia , Extratos Vegetais/imunologia , Quillaja/química , Saponinas/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/química , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Linfócitos T CD8-Positivos , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Relação Dose-Resposta Imunológica , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/efeitos adversos , Extratos Vegetais/química , Folhas de Planta/química , Saponinas de Quilaia/administração & dosagem , Saponinas de Quilaia/efeitos adversos , Saponinas de Quilaia/imunologia , Saponinas/química , Saponinas/economia , Saponinas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Equilíbrio Th1-Th2 , Vacinas Virais/administração & dosagemRESUMO
Bovine viral diarrhea virus (BVDV) infects cattle populations worldwide, causing significant economic losses though its impact on animal health. Previous studies have reported the prevalence of BVDV species and subgenotypes in cattle from the United States and Canada. We investigated the genetic diversity of BVDV strains detected in bovine serum samples from 6 different Mexican regions. Sixty-two BVDV isolates from Mexico were genetically typed based on comparison of sequences from the 5' untranslated region (5'-UTR) of the viral genome. Phylogenetic reconstruction indicated that 60 of the samples belonged to the BVDV-1 genotype and 2 to the BVDV-2 genotype. Comparison of partial 5'-UTR sequences clustered 49 samples within BVDV-1c, 8 samples within BVDV-1a, 3 samples within BVDV-1b, and 2 samples clustered with the BVDV-2a subgenotypes. Our study, combined with information previously published on BVDV field strain diversity in the United States and Canada, benefits the development of effective detection assays, vaccines, and control programs for North America.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 2/isolamento & purificação , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Variação Genética , Genótipo , México/epidemiologia , FilogeniaRESUMO
Objetivou-se descrever os aspectos clínicos e anatomopatológicos, e a identificação viral de um caso de infecção pelo vírus 'Hobi'-like (BVDV-3) em bovino do semiárido paraibano, Nordeste do Brasil. Um bovino, fêmea, três meses de idade, foi levado ao Hospital Veterinário da UFCG apresentando salivação, dificuldade de apreensão do teto, falta de apetite, fezes escuras e em pouca quantidade. Diante da piora do quadro clínico optou-se por sua eutanásia in extremis, seguida da realização da necropsia e coleta de material para histopatologia. Histologicamente, nas mucosas do trato digestivo, havia edema, degeneração balonosa, necrose e infiltrado inflamatório, que foi observado na face dorsal da língua e no seu epitélio mais profundo. A imunohistoquímica de amostras de extremidade de pavilhão auricular demonstrou marcação antigênica positiva e pela RT-PCR foi possível detectar RNA viral do BVDV no soro sanguíneo, cujo efeito citopático em células epiteliais de rim bovino da linhagem "Madin Darby bovine kidney" (MDBK) não foi observado. O sequenciamento do gene 5'NCR demonstrou que o vírus isolado estaria mais relacionado ao 'Hobi'-like (BVDV-3). Após a confirmação do diagnóstico foram coletadas amostras de soro dos 23 animais do rebanho para sorologia por ELISA indireto, sendo constatada 69,6% (16/23) de soropositividade. A identificação deste novo caso de infecção por 'Hobi'-like na Paraíba reafirma a necessidade de um monitoramento regular para BVDV na região para detecção precoce da infecção dos rebanhos e adoção de medidas eficazes de prevenção e controle.(AU)
The aim of this study was to describe the clinical and anatomopathological aspects, and the viral identification of a case of 'Hobi'-like (BVDV-3) vírus infection in cattle from the semiarid of Paraiba State, Northeastern Brazil. A female bovine, three months old, was sent to the UFCG's Veterinary Hospital presenting salivation, difficulty of ceiling seizure, lack of appetite, dark feces and in small amounts. Due to worsening of symptoms it was decided to in extremis euthanasia, followed by the necropsy and collection of material for histopathology. Histologically, in the mucous membranes of the digestive tract there were edema, ballooning degeneration, necrosis and inflammatory infiltrate, which was observed on the dorsal surface of the tongue and in its deepest epithelium. The immunohistochemical of skin biopsies of the extremity of the ear (ear notches) showed positive antigenic marking and by RT-PCR it was possible to detect viral RNA of BVDV in the serum, whose cytopathic effect in epithelial cells of bovine kidney lineage "Madin Darby bovine kidney" (MDBK)was not observed. The sequencing of the 5'NCR gene showed that the virus isolated was more related to 'Hobi'-like (BVDV-3). After confirming the diagnosis serum samples were collected from 23 animals for serology by indirect ELISA, and a seropositivity of 69.6% (16/23) was found. The identification of this new case of 'Hobi'-like infection in Paraiba reaffirms the need for regular BVDV monitoring in the region to early detection of infection in the herds and adoption of effective preventive and control measures.(AU)
Assuntos
Animais , Feminino , Bovinos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina , Infecções por Pestivirus/veterinária , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Ensaio de Imunoadsorção Enzimática/veterinária , Imuno-Histoquímica/veterinária , Pestivirus/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Serological surveys based on a planned sampling on bovine viral diarrhea virus (BVDV) infection in Brazilian cattle herds are scarce. A cross-sectional study was carried out to determine herd- and animal-level seroprevalences and to identify risk factors associated with herd-level seroprevalence for BVDV infection in the State of Paraíba, Northeastern Brazil, from September 2012 to January 2013. The state was divided into three sampling strata, and for each stratum, the prevalence of herds infected with BVDV and the prevalence of seropositive animals was estimated by a two-stage sampling survey. In total, 2443 animals were sampled from 478 herds. A virus-neutralization test was used for BVDV antibody detection. A herd was considered positive when at least one seropositive animal was detected. The herd- and animal-level prevalences in the State of Paraíba were 65.5% (95% confidence interval (CI) = 61.1-69.7%) and 39.1% (95% CI = 33.1-45.6%), respectively. The frequency of seropositive animals per herd ranged from 10 to 100% (median of 50%). The risk factors identified were as follows: more than six calves aged ≤12 months (odds ratio (OR) = 3.72; 95% CI = 2.08-6.66), animal purchasing (OR = 1.66; 95% CI = 1.08-2.55), pasture rental (OR = 2.15; 95% CI = 1.35-3.55), and presence of veterinary assistance (OR = 2.04; 95% CI = 1.10-3.79). Our findings suggest that the implementation of control and prevention measures among farmers, with the aim of preventing dissemination of the agent in the herds, is necessary. Special attention should be given to addressing the identified risk factors, such as sanitary control prior to animal purchasing and to discourage the pasture rental, as well as to encourage the vaccination in the herds.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Animais , Anticorpos Antivirais/sangue , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Brasil/epidemiologia , Bovinos , Estudos Transversais , Diarreia , Vírus da Diarreia Viral Bovina/imunologia , Prevalência , Fatores de Risco , Estudos Soroepidemiológicos , Vacinação/veterináriaRESUMO
The aim of this study was to describe the clinical and anatomopathological aspects, and the viral identification of a case of Hobi-like (BVDV-3) vírus infection in cattle from the semiarid of Paraiba State, Northeastern Brazil. A female bovine, three months old, was sent to the UFCGs Veterinary Hospital presenting salivation, difficulty of ceiling seizure, lack of appetite, dark feces and in small amounts. Due to worsening of symptoms it was decided to in extremis euthanasia, followed by the necropsy and collection of material for histopathology. Histologically, in the mucous membranes of the digestive tract there were edema, ballooning degeneration, necrosis and inflammatory infiltrate, which was observed on the dorsal surface of the tongue and in its deepest epithelium. The immunohistochemical of skin biopsies of the extremity of the ear (ear notches) showed positive antigenic marking and by RT-PCR it was possible to detect viral RNA of BVDV in the serum, whose cytopathic effect in epithelial cells of bovine kidney lineage Madin Darby bovine kidney (MDBK)was not observed. The sequencing of the 5NCR gene showed that the virus isolated was more related to Hobi-like (BVDV-3). After confirming the diagnosis serum samples were collected from 23 animals for serology by indirect ELISA, and a seropositivity of 69.6% (16/23) was found. The identification of this new case of Hobi-like infection in Paraiba reaffirms the need for regular BVDV monitoring in the region to early detection of infection in the herds and adoption of effective preventive and control measures.(AU)
Objetivou-se descrever os aspectos clínicos e anatomopatológicos, e a identificação viral de um caso de infecção pelo vírus Hobi-like (BVDV-3) em bovino do semiárido paraibano, Nordeste do Brasil. Um bovino, fêmea, três meses de idade, foi levado ao Hospital Veterinário da UFCG apresentando salivação, dificuldade de apreensão do teto, falta de apetite, fezes escuras e em pouca quantidade. Diante da piora do quadro clínico optou-se por sua eutanásia in extremis, seguida da realização da necropsia e coleta de material para histopatologia. Histologicamente, nas mucosas do trato digestivo, havia edema, degeneração balonosa, necrose e infiltrado inflamatório, que foi observado na face dorsal da língua e no seu epitélio mais profundo. A imunohistoquímica de amostras de extremidade de pavilhão auricular demonstrou marcação antigênica positiva e pela RT-PCR foi possível detectar RNA viral do BVDV no soro sanguíneo, cujo efeito citopático em células epiteliais de rim bovino da linhagem Madin Darby bovine kidney (MDBK) não foi observado. O sequenciamento do gene 5NCR demonstrou que o vírus isolado estaria mais relacionado ao Hobi-like (BVDV-3). Após a confirmação do diagnóstico foram coletadas amostras de soro dos 23 animais do rebanho para sorologia por ELISA indireto, sendo constatada 69,6% (16/23) de soropositividade. A identificação deste novo caso de infecção por Hobi-like na Paraíba reafirma a necessidade de um monitoramento regular para BVDV na região para detecção precoce da infecção dos rebanhos e adoção de medidas eficazes de prevenção e controle.(AU)
Assuntos
Animais , Feminino , Bovinos , Vírus da Diarreia Viral Bovina , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Infecções por Pestivirus/veterinária , Pestivirus/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Imuno-Histoquímica/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controleRESUMO
Bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle that occurs worldwide with substantial economic impact on beef and dairy industries. The aim of this study was to describe the diversity of BVDV subgenotypes in persistently infected (PI) animals identified in a highly productive, regularly vaccinated, dairy cattle herd presenting with reproductive failure. Serum samples were collected from all animals within the herd (n = 692) and used to detect the presence of BVDV RNA. Using reverse transcription polymerase chain reaction assay, 29 cows were identified as transiently infected, three animals (two cows and one calf) as persistently infected, and one calf as putative BVDV PI animal. The sequences of 5'UTR and/or N(pro) gene of BVDV used in phylogenetic analyses revealed that the three PI animals were infected by three different BVDV subgenotypes (BVDV-1a, BVDV-1b, and BVDV-1d). These results demonstrated that in an open dairy cattle herd, regular vaccination against BVDV by itself is not able to prevent viral circulation in the herd. Furthermore, depending on the frequency of the acquisition of heifers and/or cows for replacement, several BVDV subgenotypes may co-exist simultaneously in the same herd.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Vírus da Diarreia Viral Bovina/genética , Vacinas Virais , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Brasil , Bovinos , Indústria de Laticínios , Vírus da Diarreia Viral Bovina/classificação , Vírus da Diarreia Viral Bovina/imunologia , Feminino , Variação Genética , Genótipo , Masculino , FilogeniaRESUMO
Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 µg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 µg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Medicago sativa/genética , Medicago sativa/imunologia , Vacinas Virais/farmacologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/genética , Feminino , Cobaias , Masculino , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Resultado do Tratamento , Vacinação/veterinária , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Four experiments evaluated the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp. on reproductive performance of lactating dairy cows without (experiments 1, 2, and 3) or with previous vaccination against these diseases (experiment 4). Cows were assigned to a fixed-time AI protocol (FTAI; d -11 to 0) in all experiments, as well as AI 12 h upon estrus detection in experiment 3. Pregnancy status was determined with transrectal ultrasonography on d 30 and 71 (d 60 for experiment 3) after AI. Pregnancy loss was considered in cows pregnant on d 30 but non-pregnant on the subsequent evaluation. In experiment 1, 853 cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI (d -11) and 30 d after AI. Pregnancy loss was reduced (P = 0.03) in VAC cows compared with CON. In experiment 2, 287 cows received VAC or CON 30 d prior to (d -41) and at the beginning (d -11) of the FTAI. Pregnancy rates on d 30 and 71 were greater (P ≤ 0.03) in VAC cows compared with CON. In experiment 3, 1680 cows with more than 28 d in milk were randomly assigned to receive VAC or CON with doses administered 14 d apart, and inseminated within 15-135 d after the second dose. Pregnancy rates on d 30 and 60 were greater (P ≤ 0.02) in VAC cows compared with CON. In experiment 4, 820 cows received (REVAC) or not (CON) revaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI protocol (d -11). Pregnancy rates and loss were similar (P ≥ 0.54) between treatments. Hence, vaccinating naïve cows against BoHV-1, BVDV, and Leptospira spp. improved reproductive efficiency in dairy production systems, particularly when both doses were administered prior to AI.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Leptospira/imunologia , Leptospirose/veterinária , Vacinação/veterinária , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Brasil , Bovinos , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Inseminação Artificial/veterinária , Lactação , Análise dos Mínimos Quadrados , Leptospirose/imunologia , Leptospirose/prevenção & controle , Leptospirose/virologia , Gravidez , Distribuição Aleatória , Reprodução/imunologia , Vacinação/métodos , Vacinação/normasRESUMO
The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Doença das Mucosas por Vírus da Diarreia Viral Bovina/transmissão , Vírus da Diarreia Viral Bovina/isolamento & purificação , Desinfecção/métodos , Sêmen/virologia , Medicina Veterinária/métodos , Animais , Bovinos , Ensaio Cometa , Embrião de Mamíferos/virologia , Fertilização in vitro/métodos , Contagem de Espermatozoides , Carga ViralRESUMO
The objectives were to assess incidence of pregnancy losses, associate this outcome with immunization programs against reproductive diseases, and evaluate the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp., on reproductive efficiency of Brazilian cow-calf operations. In experiment 1, 7614 lactating Nelore cows from 18 ranches were assigned to the same estrus synchronization and fixed-time AI protocol (ESFTAI; Days -11 to 0). Pregnancy status was determined with transrectal ultrasonography on Days 30 and 120 after AI. Pregnancy loss was deemed to have occurred when cows were pregnant on Day 30 but nonpregnant on Day 120. Incidence of pregnancy loss across all ranches was 4.1%; pregnancy losses were detected (P < 0.10) in 14 ranches but not detected (P > 0.11) in four ranches. Pregnancy loss was lower (P ≤ 0.02) in ranches that vaccinated against BoHV-1, BVDV, and Leptospira spp. compared with ranches that did not vaccinate, or only vaccinated against Leptospira spp. In experiments 2 and 3, lactating Nelore cows (N = 1950 and 2793, respectively) from ranches that did not have a history of vaccinating against reproductive diseases (experiment 2), or only vaccinated against Leptospira spp. (experiment 3), were assigned to the same ESFTAI used in experiment 1. Within each ranch, cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the ESFTAI (Day -11) and 30 days after (Day 41) AI. In experiment 2, VAC cows had greater (P ≤ 0.05) pregnancy rates compared with CON on Days 30 and 120. In experiments 2 and 3, pregnancy loss was reduced (P ≤ 0.03) in primiparous VAC cows compared with CON cohorts. In experiment 4, 367 primiparous, lactating Nelore cows previously vaccinated against Leptospira spp. were assigned to the same ESFTAI used in experiment 1. Cows received VAC, or the same vaccine 30 days before (Day -41) and at the beginning (Day -11) of the ESFTAI (PREVAC). Pregnancy rates on Days 30 and 120 were greater (P ≤ 0.05) in PREVAC cows compared with VAC cows. In conclusion, pregnancy losses affected reproductive and overall efficiency of Brazilian cow-calf operations, and might be directly associated with BoHV-1, BVDV, and Leptospira spp. infections. Hence, vaccinating cows against these pathogens, particularly when both doses are administered before fixed-time AI, improved reproductive performance in Brazilian cow-calf systems.
Assuntos
Doenças dos Bovinos/prevenção & controle , Inseminação Artificial/veterinária , Reprodução/fisiologia , Vacinação/veterinária , Aborto Animal/epidemiologia , Animais , Composição Corporal , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Vírus da Diarreia Viral Bovina/imunologia , Feminino , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Leptospira/imunologia , Leptospirose/prevenção & controle , Leptospirose/veterinária , GravidezRESUMO
Bovine viral diarrhea (BVD) infection caused by bovine viral diarrhea virus (BVDV), a Pestivirus of the Flaviviridae family, is an important cause of morbidity, mortality and economical losses in cattle worldwide. E2 protein is the major glycoprotein of BVDV envelope and the main target for neutralising antibodies (NAbs). Different studies on protection against BVDV infection have focused on E2, supporting its putative use in subunit vaccines. A truncated version of type 1a BVDV E2 (tE2) expressed in mammalian cells was used to formulate an experimental oleous monovalent vaccine. Immunogenicity was studied through immunisation of guinea pigs and followed by trials in cattle. Calves of 8-12 months were vaccinated, twice with a 4 week interval, with either a tE2 subunit vaccine (n = 8), a whole virus inactivated vaccine (n = 8) or left untreated as negative control group (n = 8). Four weeks after the last immunisation the animals were experimentally challenged intranasally with a non-cythopathic BVDV strain. Following challenge, BVDV was isolated from all unvaccinated animals, while 6 out of 8 animals vaccinated with tE2 showed complete virological protection indicating that the tE2 vaccine presented a similar performance to a satisfactory whole virus inactivated vaccine.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Doenças dos Bovinos/prevenção & controle , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/efeitos adversos , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Doenças dos Bovinos/imunologia , DNA Viral/genética , DNA Viral/imunologia , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/efeitos adversos , Glicoproteínas/genética , Glicoproteínas/imunologia , Cobaias , Testes de Neutralização , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologia , Proteínas do Envelope Viral/efeitos adversos , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 µg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 µg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant.
Assuntos
Antígenos Virais/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Nicotiana/metabolismo , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Agrobacterium , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/biossíntese , Antígenos Virais/genética , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Bovinos , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Feminino , Engenharia Genética/métodos , Cobaias , Testes de Neutralização , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genéticaRESUMO
Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.
Assuntos
Antígenos Virais , Vírus da Diarreia Viral Bovina/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas do Envelope Viral , Vacinas Virais , Animais , Antígenos Virais/análise , Antígenos Virais/genética , Antígenos Virais/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Células CHO , Bovinos , Cricetinae , Cricetulus , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vacinas de Produtos Inativados/imunologia , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologiaRESUMO
Our objective was to determine the prevalence of serum antibodies to bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea (BVD) virus in beef cattle in Uruguay. A random sample of 230 herds selected with probability proportional to population size based on the number of cattle was chosen from a list frame of all registered livestock farms as of June 1999. Sera from up to 10 heifers, cows and bulls (up to 30 sera total per herd) were collected on selected farms between March 2000 and March 2001 and evaluated by means of enzyme-linked immunosorbent assays (ELISAs). Overall, 6358 serum samples were evaluated. We also collected data on previous diagnosis of BHV-1 or BVD infections and on the use of vaccines against these agents. The estimated prevalence of exposure to BHV-1 and BVD at the herd level for the Uruguayan beef population was 99% and 100%, respectively. Approximately 37% of beef cattle in Uruguay have been exposed to BHV-1 and 69% to BVD virus. Only 3% of beef herds in Uruguay regularly (typically, annually) use vaccines against either of these agents.