Development and Evaluation of an Indirect ELISA: Serological Survey to Detect Specific Antibodies to Bovine Herpesvirus 4

ABSTRACTThe aim of this work was to develop and evaluate an indirect enzyme-linked immunosorbent assay (ELISA) and made a serological screening for specific antibodies to BoHV-4. Bovine serum samples were collected from different Brazilian states and evaluated for the presence of antibodies for BoHV-4 and BoHV-1. The serological results obtained showed that the indirect ELISA assay could be applied for the detection of specific antibodies for BoHV-4. The ELISA test allowed concluding that BoHV-4 is present in bovines in every Brazilian state from which serum samples were collected. The ELISA assay here standardized proved to be useful for the epidemiological studies and showed a positivity range from 1.8 to 66% in Brazil.

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.

Occurrence of bovine herpesvirus type 4 DNA in Argentinean Holstein cattle from Santiago del Estero, Argentina / Ocorrência de DNA de herpesvírus bovino tipo 4 em gado Holstein argentino de Santiago del stero, Argentina

and from cattle with a variety of clinical signs. The pathogenic role of BoHV-4 remains unclear and it is unknown whether the virus acts as a primary pathogen or whether it facilitates secondary infections After natural or experimental infections, BoHV-4 can establish latency, mainly in cells of the monocyte/macrophage linage. Latent virus can be reactivated after glucocorticoid treatment or by stress factors. In 2007, BoHV-4 was isolated for the first time in Argentina, from samples of bovine abortions. In the present study, we used viral isolation, nested PCR and restriction endonuclease analysis (REA) to investigate the presence of BoHV-4 in bovine leukocytes from a single herd of dairy cattle with reproductive problems. In this work, we demonstrated that BoHV-4 genome is present in the leukocytes of a high proportion (63.4%) of animals, probably in a latent or persistent state. BoHV-4 was isolated from one out of eleven peripheral blood leukocyte (PBL) samples. By REA we demonstrated the existence of genomic variation among the strains circulating in this particular herd. Furthermore, all PBL samples evaluated in this study differed from the American prototype strain, DN 599. Overall, this work demonstrated that BoHV-4 is present in the leukocyte fraction of dairy cattle and that viral strains present in this herd are genetically divergent. Although BoHV-4 was detected in a herd with a background of reproductive disorders, it is not possible to conclude that the virus is the primary responsible for these conditions.

O herpesvírus bovino tipo 4 (BoHV-4) é um gama-herpesvírus que foi isolado de animais aparentemente saudáveis e de gado com uma variedade de sinais clínicos. O papel patogênico do BoHV-4 ainda não está claro e não se sabe se o vírus age como um patógeno primário ou se facilita infecções secundárias. Depois de infecções naturais ou experimentais, BoHV-4 pode estabelecer latência, principalmente nas células dos linhagens de monócitos/macrófagos. O vírus latente pode ser reativado após o uso de glicocorticóides ou por fatores de estresse. Em 2007, o BoHV-4 foi isolado pela primeira vez na Argentina, a partir de amostras de abortos bovinos. No presente estudo, utilizou-se o isolamento viral, nested PCR e análise com endonucleases de restrição (REA) para investigar a presença de BoHV4 em leucócitos de bovinos provenientes de um único rebanho de gado leiteiro com problemas reprodutivos. Neste trabalho, demonstramos que o genoma do BoHV-4 está presente nos leucócitos em uma elevada proporção (63,4%) dos animais, provavelmente em um estado latente ou persistente. BoHV-4 foi isolado de uma de cada onze amostras de leucócitos no sangue periférico (PBL). Por REA nós demonstramos a existência de variações genômicas entre as estirpes circulantes deste rebanho particular. Além disso, todas as amostras de PBL avaliados neste estudo diferiram da estirpe protótipo Americano, DN 599. Em geral, este estudo demonstrou que o BoHV-4 está presente na fração leucocitária do gado leiteiro e que as estirpes virais presentes neste rebanho são geneticamente divergentes. Embora que BoHV-4 foi detectado em um rebanho com história de distúrbios reprodutivos, não é possível concluir que o vírus é o principal responsável por estas condições.