Resumo

Bovine viral diarrhea virus (BVDV) is a productive and reproductive virus with a high global economic impact on dairy production systems. We investigated the prevalence of BVDV at the herd and individual levels in most dairy-producing regions of Brazil. The frequency of BVDV at the herd level was investigated using quantitative reverse transcription polymerase chain reaction (RT-qPCR) in bulk milk tanks monitored at 289 dairy farms between August 2020 and January 2022. Among these farms, 68 production systems were selected to investigate the prevalence of persistently infected (PI) animals using two antigen-enzyme-linked immunosorbent assays (Ag-ELISAs), at 21-d intervals, from ear-notch samples. In total, 2,902 RT-qPCR and 23,466 Ag-ELISAs were performed. At the herd level, 23.87% (69/289) of dairy farms were considered infected, presenting at least one qPCR test positive. At the individual level, 41.2% (28/68) of the subgroup of selected farms had at least one animal positive in the Ag-ELISA test. Association between tests allowed the classification of farms into the following four categories: level 0, negative for both tests (41.2%, 28/68); level 1, RT-qPCR positive and Ag-ELISA negative (17.6%, 12/68); level 2, RT-qPCR negative and Ag-ELISA positive (13.2%, 9/68); and level 3, positive for both tests (27.9%, 19/68). Multiple correspondence analysis (MCA) suggested a possible association between BVDV positivity and large farms, average daily milk production (herd), breed, and somatic cell counts. The confinement and intensification of animals from different categories, and use of artificial bedding are associated with BVDV infection. Using waste or bulk tank milk to fed calves was also a risk factor for BVDV positivity in RT-qPCR and Ag-ELISA. Despite the use of reproductive vaccines by most producers, their use seems to be associated with BVDV-positive farms. This study presented the epidemiological frequencies of BVDV at the individual and herd levels in the Campos Gerais Paranaense region. The region ranks among the top milk-producing areas in Brazil. Additionally, the association between BVDV tests and farm characteristics indicated the farm risk for BVDV and guides specific control programs.

O vírus da diarreia viral bovina (BVDV) é um vírus que afeta a produção e reprodução com alto impacto econômico global nos sistemas de produção de leite. Nosso objetivo foi investigar a prevalência do BVDV em rebanhos e indivíduos na região de maior produção de leite do Brasil. A frequência do BVDV nos rebanhos foi investigada usando reação em cadeia da polimerase com transcrição reversa quantitativa (RT-qPCR) em tanques de leite em 289 fazendas leiteiras, monitoradas entre agosto de 2020 e janeiro de 2022. Dentre esses rebanhos, 68 sistemas de produção foram selecionados para investigar a prevalência de animais persistentemente infectados (PI) usando dois testes imunoenzimáticos para a detecção do antígeno (Ag-ELISAs), em intervalos de 21 dias, a partir de amostras de tecido auricular. No total, foram realizados 2.902 RT-qPCR e 23.466 Ag-ELISAs. Na investigação dos rebanhos, 23,9% (69/289) das propriedades leiteiras foram consideradas infectadas, apresentando pelo menos um teste qPCR positivo. Na pesquisa de animais Pis, 41,2% (28/68) das fazendas selecionadas tiveram pelo menos um animal positivo no teste Ag-ELISA. A associação entre os testes permitiu classificar as propriedades em quatro categorias: nível 0, negativo para ambos os testes (41,2%, 28/68); nível 1, RT-qPCR positivo e Ag-ELISA negativo (17,6%, 12/68); nível 2, RT-qPCR negativo e Ag-ELISA positivo (13,23%, 9/68); e nível 3, positivo para ambos os testes (27,9%, 19/68). A análise de correspondência múltipla (MCA) sugeriu associação entre positividade para BVDV e grandes propriedades, produção média diária de leite do rebanho, raça e contagem de células somáticas. O confinamento e a intensificação de animais de diferentes categorias e o uso de camas artificiais estão associados à infecção pelo BVDV. O uso de leite residual ou a granel também foi um fator de risco para positividade para BVDV em RT-qPCR e Ag-ELISA. Apesar da utilização de vacinas reprodutivas pela maioria dos produtores, a sua utilização parece estar associada a explorações positivas para BVDV. Este estudo apresenta as frequências epidemiológicas do BVDV nos rebanhos e indivíduos de rebanhos da região dos Campos Gerais Paranaense. A região está entre as principais áreas produtoras de leite do Brasil. Além disso, a associação entre os testes de BVDV e as características da fazenda indica o risco da fazenda para o BVDV e orienta programas de controle específicos.

Resumo

This study determined the association between biosecurity practices and the status of bovine viral diarrhea virus (BVDV) in dairy production systems. Approximately 280 herds were screened for BVDV virus detection. Following the screening, 68 herds were selected to identify individual BVDV PI animals using an ear notch biopsy and ELISA-antigen. All offspring of the last generation were tested, and the maternal lineage of positive cases was examined. A questionnaire on BVDV biological risk assessment was completed. A multiple correspondence analysis (MCA) was conducted to determine the association between herds with or without BVDV circulation and biosecurity practices. The MCA revealed that farms with virus circulation lacked knowledge about the disease and wrongly perceived their herds as protected, while farms without virus circulation were aware of the disease but considered their herds unprotected. Vaccination practices differed between positive and negative herds, with positive herds using vaccines only for reproductive diseases and negative herds vaccinating for respiratory and reproductive issues. Biosecurity practices such as frequent visitation, contact between animals of different ages, and annual introduction of new animals were linked to viral circulation, while virus-free herds implemented measures like controlled visitation, no contact between different age groups, and quarantine. Lastly, herds with virus circulation acquired pregnant females without prior testing. This study emphasized the crucial role of biosecurity practices in controlling BVDV in dairy herds. It highlighted the sharper risk perception and better application of biosecurity practices in negative herds compared to BVDV-positive herds.

Esta pesquisa teve como objetivo associar as práticas de biosseguridade com a circulação do vírus BVDV em sistemas de produção leiteiros. Inicialmente, 270-290 sistemas foram selecionados para detecção do vírus entre janeiro de 2020 e janeiro de 2023 por meio da técnica de Reação em Cadeia da Polimerase em Tempo Real (RT-qPCR em tempo real). Após essa etapa, 68 rebanhos foram selecionados para identificação de animais persistentemente infectados (PI) por meio da análise individual do fragmento auricular usando o método ELISA-antígeno. Todos os animais nascidos na última geração, fêmeas que deram à luz bezerros machos ou natimortos, foram testados incluindo a pesquisa na geração de mães e avós maternas em casos positivos. Um questionário de análise de risco biológico para o BVDV foi aplicado a esse grupo de fazendas. Para identificar associações entre rebanhos com ou sem circulação do BVDV e práticas de biosseguridade, foi usada a análise de correspondência múltipla (MCA). A análise mostrou associação entre a falta de conhecimento sobre a doença e a percepção de que o rebanho está protegido contra o BVDV em fazendas com circulação do vírus. Os rebanhos positivos para Diarreia Viral Bovina reportaram o uso vacinas apenas para doenças reprodutivas, enquanto os rebanhos negativos reportaram o uso vacinas para problemas respiratórios e reprodutivos, sugerindo possíveis diferenças nos protocolos de vacinação e na eficácia das vacinas no Brasil. Práticas de biosseguridade como visitas frequentes, contato entre animais de diferentes idades e a introdução anual de novos animais, foram associadas à circulação viral, enquanto os rebanhos livres de vírus adotaram medidas como políticas de visitação e controle, ausência de contato entre os bovinos de diferentes idades e quarentena. Além disso, os sistemas de produção positivos adquiriram fêmeas prenhes sem testar o animal previamente. Assim, este estudo destaca aspectos essenciais para o controle do vírus da Diarreia Viral Bovina em rebanhos leiteiros, identificando que gestores de rebanhos negativos possuem maior percepção de risco associada à implementação de práticas de biosseguridade, em contraste comos rebanhos positivos para o BVDV.

Resumo

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre-and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the x² or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.