PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P< 0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.

• Nuclear maturation rate oocytes bovine herpesvirus 1 BoHV-1 IBR maturation cattle pathology

Abstract in Portuguese:

Herpesvírus bovino 1 (BoHV 1) é um importante patógeno bovino, responsável por causar doenças respiratórias e falhas reprodutivas. A presença do BoHV-1 em sistema de produção in vitro de embriões afeta a fertilização, a maturação e o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a capacidade de desenvolvimento de ovócitos oriundos de vacas infectadas naturalmente sem histórico reprodutivo. Além disso, este estudo investigou a presença do DNA viral em Complexos Cumulus Ooforus (COCs). Os tratamentos foram definidos a partir do título de anticorpos detectados pelos ensaios de soroneutralização, sendo estabelecidos três grupos: animais soronegativos (título menor do que 2), título baixo (2 a 8) e animais com título maior ou igual a 16. Os COCs foram obtidos de 15 doadoras durante 22 sessões de aspiração folicular guiada por ultrassom. A extração do DNA foi realizada em um pool de COCs de todas as aspirações de uma mesma doadora e no sangue total para a realização das reações de Nested-PCR. Para avaliação da taxa de maturação nuclear, foram selecionados para o cultivo in vitro somente os COCs com camada compacta de células do cumulus, zona pelúcida íntegra e citoplasma homogêneo. Após 24 horas de cultivo, os ovócitos foram fixados e corados em lâmina para a avaliação do estádio do ciclo celular meiótico. Os ovócitos que apresentaram configuração cromossômica em metáfase II foram considerados como tendo alcançado a maturação nuclear. Verificou-se comprometimento na taxa de maturação nuclear ovocitária (P<0.05) nos animais de título maior ou igual a 16 (50%). No entanto, não houve influência do título viral na taxa de maturação de ovócitos bovinos em animais que apresentaram titulação baixa (62,2%) quando comparados com o grupo controle (76,7%). O DNA viral não foi identificado nas amostras de sangue, mas foi detectado no pool de COCs de três doadoras soropositivas. Diante dos resultados encontrados conclui-se que vacas infectadas naturalmente pelo vírus BoHV-1 apresentam comprometimento na taxa de maturação nuclear, dependendo do grau de titulação de anticorpos contra o vírus. Ademais, o DNA viral pode estar presente em COCs contrariando a hipótese de que animais sorologicamente positivos e sem histórico de sintomatologia clínica oferecem risco negligível de transmissão do BoHV-1 por COCs.

• Taxa de maturação nuclear ovócitos vacas herpesvírus bovino 1 BoHV-1 IBR maturação bovinos patologia

Abstract in English:

ABSTRACT.- Weiss M., Anziliero D., Martins M., Weiblen R. & Flores E.F. 2016. Safety and immunogenicity of a glycoprotein E gene-deleted bovine herpesvirus 1 strain as a candidate vaccine strain. Pesquisa Veterinária Brasileira 36(11):1067-1074. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Av. Roraima 1000, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com A glycoprotein E-deleted Brazilian bovine herpesvirus 1 (BoHV-1gE&#8710;) was tested regarding to safety and immunogenicity. Intramuscular inoculation of young calves with a high virus dose did not result in clinical signs or virus shedding during acute infection or after dexamethasone administration. Calves vaccinated once IM (group I) or subcutaneously (group II) with live BoHV-1gE&#916; or twice with inactivated virus plus aluminum hydroxide (group IV) or MontanideTM (group V) developed VN titers of 2 to 8 (GMT:2); 2 to 4 (GMT:1.65); 2 to 16 (GMT:2.45) and 2 to 128 (GMT:3.9), respectively. All BoHV-1gE&#916; vaccinated calves remained negative in an anti-gE ELISA. Lastly, six young calves vaccinated with live BoHV-1gE&#8710; and subsequently challenged with a virulent BoHV-1 strain shed less virus and developed only mild and transient nasal signs comparing to unvaccinated calves. Thus, the recombinant BoHV-1gE&#8710; is safe and immunogenic for calves and allows for serological differentiation by a gE-ELISA test.

• BoHV-1 vaccine vacinas

Abstract in Portuguese:

RESUMO.- Weiss M., Anziliero D., Martins M., Weiblen R. & Flores E.F. 2016. Safety and immunogenicity of a glycoprotein E gene-deleted bovine herpesvirus 1 strain as a candidate vaccine strain. [Atenuação e imunogenicidade de uma cepa recombinante do herpesvírus bovino tipo 1 defectiva na glicoproteína E.] Pesquisa Veterinária Brasileira 36(11):1067-1074. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Av. Roraima 1000, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com Um isolado brasileiro de herpesvírus bovino tipo 1, contendo uma deleção na glicoproteína E (gE - BoHV-1gE&#8710;) foi submetido a testes para avaliar a sua segurança e imunogenicidade em bovinos. Bezerros foram submetidos à inoculação intramuscular com uma alta dose viral e não demonstraram sinais clínicos ou excreção viral durante a fase aguda ou após tentativa de reativação viral pela administração de dexametasona. Bezerros que receberam uma dose do vírus vivo, contendo a deleção na gE, pela via intramuscular (grupo I) ou pela via subcutânea (grupo II) ou duas doses do vírus inativado utilizando o adjuvante hidróxido de alumínio (grupo IV) ou MontanideTM (grupo V), desenvolveram títulos de anticorpos neutralizantes de 2 a 8 (GMT: 2); 2 a 4 (GMT: 1,65); 2 a 16 (GMT: 2,25) e de 2 a 128 (GMT: 3,9), respectivamente. Todos os bezerros vacinados se mantiveram soronegativos quando utilizado um kit ELISA específico para a gE. Para o teste de segurança, seis bezerros foram vacinados com o vírus vivo BoHV-1gE&#916;, sendo estes posteriormente desafiados com uma cepa virulenta de BoHV-1. Estes bezerros excretaram menos vírus e desenvolveram apenas sinais clínicos moderados e transitórios quando comparados com dados coletados de quatro animais não-vacinados. Com base nestes resultados, podemos confirmar que o vírus do BoHV-1 que contém a deleção na gE (BoHV-1gE&#916;) é seguro e suficientemente imunogênico para bezerros e permite a diferenciação sorológica entre os animais vacinados e infectados perante a a um teste ELISA commercial específico para a gE.

Abstract in English:

ABSTRACT.- Oliveira S.A.M., Brum M.C.S., Anziliero D., Dellagostin O., Weiblen R. & Flores E.F. 2013. Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and use in an ELISA for gE antibodies. Pesquisa Veterinária Brasileira 33(1):41-46. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Avenida Roraima 1000, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

• BoHV-5 bovine herpesvirus vaccine herpesvírus bovino vacina diferencial

Abstract in Portuguese:

RESUMO.- Oliveira S.A.M., Brum M.C.S., Anziliero D., Dellagostin O., Weiblen R. & Flores E.F. 2013. Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and use in an ELISA for gE antibodies. [Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE.] Pesquisa Veterinária Brasileira 33(1):41-46. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Avenida Roraima 1000, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 – que compartilha uma alta identidade com a gE do BoHV-5 – foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.

Abstract in English:

ABSTRACT.- Arruda L.P., Nakazato L., Dutra V., Lemos R.A.A., Nogueira A.P.A., Cruz R.A.S., Pescador C.A. & Colodel E.M. 2010. [Molecular detection of bovine herpesvirus 1 and 5 in formalin-fixed, paraffin-embedded samples from cattle with neurological disease.] Detecção molecular de herpesvírus bovino 1 e 5 em amostras de encéfalo conservadas em formol e emblocadas em parafina provenientes de bovinos com doença neurológica. Pesquisa Veterinária Brasileira 30(8):646-650. Departamento de Clínica Médica Veterinária, Faculdade de Agronomia e Medicina Veterinária, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa s/n, Bairro Coxipó, Cuiabá, MT 78068-900, Brazil. E-mail: laura.peixoto@gmail.com Bovine herpesvirus (BoHV) is an important cause of neurological disease in cattle in the Midwest Brazil. The application of molecular diagnostic techniques represents an important contribution for the study of BoHV. This paper describes the detection of BoHV-5 and BoHV-1 by a specific multiplex PCR assay in 76 paraffin-embedded samples from central nervous system (CNS) of cattle with neurological disorders. The samples were divided into 2 groups according to the histological features: Group 1 was composed of 40 cases of necrotizing meningoencephalitis (characteristic of BoHV infection), and Group 2 was composed of 36 cases of nonspecific nonsuppurative meningoencephalitis. Positive results for BoHV-5 accounted for 40% of the samples in the group 1 and 33% in the group 2. No detection of BoHV-1 was recorded.

• Bovine herpesvirus 1 bovine herpesvirus 5 BoHV-1 BoHV-5 herpesvírus bovino 1 herpesvírus bovino 5

Abstract in Portuguese:

RESUMO.- Arruda L.P., Nakazato L., Dutra V., Lemos R.A.A., Nogueira A.P.A., Cruz R.A.S., Pescador C.A. & Colodel E.M. 2010. [Molecular detection of bovine herpesvirus 1 and 5 in formalin-fixed, paraffin-embedded samples from cattle with neurological disease.] Detecção molecular de herpesvírus bovino 1 e 5 em amostras de encéfalo conservadas em formol e emblocadas em parafina provenientes de bovinos com doença neurológica. Pesquisa Veterinária Brasileira 30(8):646-650. Departamento de Clínica Médica Veterinária, Faculdade de Agronomia e Medicina Veterinária, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa s/n, Bairro Coxipó, Cuiabá, MT 78068-900, Brazil. E-mail: laura.peixoto@gmail.com A infecção por herpesvírus bovino (BoHV) é uma das principais causas de doença neurológica em bovinos na região Centro-Oeste do Brasil. O uso de técnicas moleculares de diagnóstico representa uma contribuição importante para o estudo dessa doença. Este trabalho descreve o uso de uma técnica específica de PCR multiplex para identificar BoHV-5 e BoHV-1 em 76 amostras de encéfalo de bovinos fixadas em formol e incluídas em parafina. Com base nas alterações histológicas, as amostras foram separadas em 2 grupos: o Grupo 1 era composto de 40 amostras de bovinos com meningoencefalite necrosante característica da infecção por BoHV; no Grupo 2 estavam 36 amostras de casos com encefalite não-supurativa inespecífica. Identificação de BoHV-5 foi constatada em 40% das amostras do grupo 1 e em 33% das amostras do grupo 2. Não houve amplificação de DNA de BoHV-1 em nenhuma amostra.

Abstract in English:

ABSTRACT.- Holz C.L., Cibulski S.P., Teixeira T.F., Batista H.B.C.R., Dezen D., Campos F.S., Varela A.P.M. & Roehe P.M. 2010. Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5. Pesquisa Veterinária Brasileira 30(7):515-522. Instituto de Pesquisas Veterinárias Desidério Finamor, Fepagro Saúde Animal, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@gmail.com The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains “Los Angeles” and “EVI123/98”; BoHV-1.2a strain “SV265/96”) and three type 5 viruses (BoHV-5a strain “EVI88/95”; BoHV-5b strain “A663” and BoHV-5c “ISO97/95”). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37%) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7% to 81.7%, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41%. These results indicate that one hour incubation SN tests against single viruses, as performed here, may display a significantly low sensitivity (p=0.05); performing SN tests against a number of different viruses may increase considerably SN sensitivity. Furthermore, the choice of virus used for challenge is critical in SN tests. In addition, sera from different geographic regions may give rise to disagreeing results with different strains of BoHV-1 and BoHV-5. This might be particularly relevant for control programs and in international trade, were maximum sensitivity should be targeted.

• BoHV-1 BoHV-5 bovine herpesvirus 1 and 5 herpesvírus bovinos 1e 5

Abstract in Portuguese:

RESUMO.- Holz C.L., Cibulski S.P., Teixeira T.F., Batista H.B.C.R., Dezen D., Campos F.S., Varela A.P.M. & Roehe P.M. 2010. Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5. [Soroneutralização com diferentes tipos e subtipos de herpesvírus bovinos 1e 5.] Pesquisa Veterinária Brasileira 30(7):515-522. Instituto de Pesquisas Veterinárias Desidério Finamor, Fepagro Saúde Animal, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@gmail.com O teste de soroneutralização (SN) é o método padrão para a mensuração de anticorpos neutralizantes para herpesvírus bovinos. Entretanto, com as subdivisões propostas destes agentes em tipos e subtipos, a definição de qual amostra utilizar como virus de desafio à SN pode ser difícil. Em vista disso, este estudo foi realizado para re-avaliar a sensibilidade de testes de SN utilizando diferentes tipos e subtipos de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) como amostras de desafio. Soros bovinos (n=810) foram coletados de duas regiões geográficas distintas e testados frente a amostras do tipo 1 (BoHV-1.1: amostras “Los Angeles” e “EVI123/98”, BoHV-1.2a: amostra “SV265/96”) e três amostras do tipo 5 (BoHV-5a: “EVI88/95”; BoHV-5b: “A663” e BoHV-5c “ISO97/95”). Os testes de SN foram realizados com incubação de 1 hora a 37ºC da mistura soro-vírus, frente a 100 doses infectantes para 50% dos cultivos celulares (DICC50) de cada um dos vírus. A sensibilidade da SN variou grandemente em função do vírus utilizado no teste. A maior sensibilidade (327 soros positivos/810 soros testados; 40.37%) foi alcançada quando os resultados positivos frente aos seis diferentes vírus foram somados. Nenhuma associação foi detectada entre determinado tipo/subtipo de vírus e a sensibilidade do teste. Quando resultados positivos frente a cada vírus foram considerados isoladamente, a sensibilidade da SN variou entre 41,7% a 81,7%, dependendo do vírus de desafio e da região geográfica de origem das amostras de soro. Variação foi detectada mesmo quando as amostras de desafio pertenciam a um mesmo subtipo; a discrepância entre os resultados positivos atingiu até 41%. Estes resultados indicam que testes de SN contra amostras isoladas de vírus podem apresentar uma sensibilidade notadamente baixa; o emprego de diferentes amostras de vírus de desafio pode aumentar consideravelmente a sensibilidade da prova. Além disso, a escolha da amostra de vírus para a realização do teste é crítica. Outro achado importante é que sorors de diferentes regiões geográficas podem dar resultados discordantes frente a diferentes amostras de BoHV-1 e BoHV-5. Estes achados são particularmente relevantes para programas de controle destas infecções e para o comércio internacional, onde a sensibilidade deve ser maximizada.

Abstract in English:

ABSTRACT.- Weiss M., Vogel F.S.F., Martins M., Weiblen R., Roehe P.M., Franco A.C. & Flores E.F. 2010. Genital immunization of heifers with a glycoprotein E-deleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate. Pesquisa Veterinária Brasileira 30(1):42-50. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: eduardofurtadoflores@gmail.com Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 106.9TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 107.6TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV-56/90, 107.1TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

• Bovine herpesvirus BoHV-1.2 vulvovaginitis

Abstract in Portuguese:

RESUMO.- Weiss M., Vogel F.S.F., Martins M., Weiblen R., Roehe P.M., Franco A.C. & Flores E.F. 2010. Genital immunization of heifers with a glycoprotein E-deleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate. [Imunização genital de bezerras com uma cepa recombinante do herpesvírus bovino tipo 1 defectiva na glicoproteína E confere proteção frente a desafio com um isolado virulento.] Pesquisa Veterinária Brasileira 30(1):42-50. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: eduardofurtadoflores@gmail.com A infecção genital de novilhas ou vacas soronegativas pelo herpesvírus bovino tipo 1.2 (BoHV-1.2) pode resultar em vulvovaginite e infertilidade temporária. As vacinas atenuadas ou inativadas administradas pela via parenteral freqüentemente conferem proteção incompleta frente a desafio pela via genital. Este estudo relata uma avaliação da resposta imunológica e proteção conferida pela vacinação genital de bezerras soronegativas com uma cepa recombinante do BoHV-1 defectiva na glicoproteína E (SV265gE-). Um grupo de seis bezerras foi vacinado com a cepa SV265gE- (0,2mL contendo 106,9TCID50) na submucosa da vulva (grupo IV); quatro bezerras foram vacinadas pela via intramuscular (IM; dose 107,6TCID50) e quatro bezerras permaneceram como controles não-vacinadas. As bezerras vacinadas pela via IV apresentaram edema e hiperemia leve e transitório na vulva e excretaram vírus em títulos baixos por alguns dias após a vacinação, porém uma bezerra soronegativa mantida em contato não soroconverteu. Administração de dexametasona pela via intravenosa no dia 70pv (0,5mg/kg) em duas bezerras vacinadas pela via IV não resultou em excreção viral, recrudescência clínica ou soroconversão. No dia 70pv, as bezerras vacinadas e as controle foram desafiadas pela inoculação genital da cepa de BoHV-1.2 altamente virulenta SV-56/90 (107.1TCID50/animal). Após o desafio, excreção viral nas secreções genitais das bezerras controle foi detectada por 8,2 dias (8-9); no grupo IM durante 6,2 dias (4-8 dias) e durante 5,2 dias (5-6) nas bezerras do grupo IN. As bezerras do grupo controle desenvolveram vulvo-vaginite moderada (2/4) a severa (2/4) que duraram entre 9 e 13 dias (x: 10,7 dias). A doença se caracterizou por edema vulvar, congestão vulvo-vestibular, formação de vesículas/pústulas que coalesceram, erosões, placas fibrino-necróticas e exsudato fibrino-purulento. As bezerras do grupo IM desenvolveram lesões genitais leves (1/3) a moderadas (3/4), com duração de 10 a 12 dias (x: 10,7 dias). No grupo IV, as bezerras desenvolveram vulvovaginite leve e transitória (3/4) ou lesões moderadas (1/4), com duração de 4 a 8 dias (x: 5,5 dias). O exame clínico desses animais após o desafio demonstrou que a vacinação, independentemente da via de administração, conferiu proteção e que, a vacinação IV mostrou-se mais efetiva na redução da severidade e duração da doença clínica. A vacinação IV também determinou uma redução significativa no período de excreção viral após desafio, em comparação com os grupos controle e IM. Esses resultados demonstram que a cepa SV265gE- administrada pela via IV confere proteção satisfatória frente a desafio local com um isolado heterólogo de BoHV-1 altamente virulento. Além disso, a cepa vacinal é atenuada para vacinação IV em baixos títulos e não é reativada facilmente após administração de dexametasona. Assim, a utilização da cepa recombinante para imunização genital pode se constituir em alternativa para prevenir a infecção e doença reprodutiva associada com o BoHV-1.

Abstract in English:

Abstract.- Silva A.D., Esteves P.A., Dezen D., Oliveira A.P., Spilki F.R., Campos F.S., Franco A.C. & Roehe P.M. 2009. Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine. Pesquisa Veterinária Brasileira 29(7):545-551. Instituto de Pesquisas Veterinárias Desidério Finamor, Fepagro Saúde Animal, Estrada do Conde 6000, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@gmail.com Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 107.0 fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

• Bovine herpesvirus type 1 BoHV-1 recombinant vaccine inactivated vaccine

Abstract in Portuguese:

Abstract.- Silva A.D., Esteves P.A., Dezen D., Oliveira A.P., Spilki F.R., Campos F.S., Franco A.C. & Roehe P.M. 2009. Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine. Pesquisa Veterinária Brasileira 29(7):545-551. Instituto de Pesquisas Veterinárias Desidério Finamor, Fepagro Saúde Animal, Estrada do Conde 6000, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@gmail.com Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 107.0 fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

Abstract in English:

ABSTRACT.- Dias J.A., Alfieri, A.A., Medici, K.C., Freitas, J.C., Ferreira-Neto J.S. & Müller E.E. 2008. [Risk factors for bovine herpesvirus 1 infection in cattle herds in the West region of Parana State.] Fatores de risco associados à infecção pelo herpesvírus bovino 1 em rebanhos bovinos da região Oeste do Estado do Paraná. Pesquisa Veterinária Brasileira 28(3):161-168. Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina. Rodovia Celso Garcia Cid 380, Cx. Postal 6001, Londrina, PR 86051-990, Brazil. E-mail: muller@uel.br The aim of this study was to determine the prevalence of positive herds and to identify the risk factors for BoHV-1 infection in breeding cattle herds in the Western region of Parana State. The statistic delineation, the serum samples and information regarding the selected farms, were the same employed in the study of bovine brucellosis for Parana State in the context of National Program for Control and Eradication of Brucellosis and Tuberculosis. The sampling was performed in two stages. Blood samples were collected from 1930 females aged ³24 months from 295 non vaccinated herds. Serum samples were tested for antibodies against BoHV-1 using an indirect ELISA. The epidemiological questionnaire was applied on all the selected farms and aimed to obtain epidemiological data. Hundred ninety of the 295 herds were positive for BoHV-1, presenting prevalence of positive herds of 64.41% [58.65-69.87%]. The number (³23) of females aged 24 months (OR=2.22; IC: 1.09-4.51), purchased cattle (OR=2.68; IC: 1.48-4.82), use of common grass (OR=5.93; IC: 1.31-26.82), history of abortion in the last 12 months (OR=2.37; IC: 1.09-5.16) and presence of wildlife animals (OR=8.86; IC: 1.11-70.73) were identified as risk factors for BoHV-1 infection in the multivariate logistic regression. These results indicate that BoHV-1 infection is widespread in the studied region and that factors related to the herd characteristic and management are associated with the infection.

• Prevalence epidemiology risk factors bovine herpesvirus 1 indirect ELISA

Abstract in Portuguese:

ABSTRACT.- Dias J.A., Alfieri, A.A., Medici, K.C., Freitas, J.C., Ferreira-Neto J.S. & Müller E.E. 2008. [Risk factors for bovine herpesvirus 1 infection in cattle herds in the West region of Parana State.] Fatores de risco associados à infecção pelo herpesvírus bovino 1 em rebanhos bovinos da região Oeste do Estado do Paraná. Pesquisa Veterinária Brasileira 28(3):161-168. Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina. Rodovia Celso Garcia Cid 380, Cx. Postal 6001, Londrina, PR 86051-990, Brazil. E-mail: muller@uel.br The aim of this study was to determine the prevalence of positive herds and to identify the risk factors for BoHV-1 infection in breeding cattle herds in the Western region of Parana State. The statistic delineation, the serum samples and information regarding the selected farms, were the same employed in the study of bovine brucellosis for Parana State in the context of National Program for Control and Eradication of Brucellosis and Tuberculosis. The sampling was performed in two stages. Blood samples were collected from 1930 females aged ³24 months from 295 non vaccinated herds. Serum samples were tested for antibodies against BoHV-1 using an indirect ELISA. The epidemiological questionnaire was applied on all the selected farms and aimed to obtain epidemiological data. Hundred ninety of the 295 herds were positive for BoHV-1, presenting prevalence of positive herds of 64.41% [58.65-69.87%]. The number (³23) of females aged 24 months (OR=2.22; IC: 1.09-4.51), purchased cattle (OR=2.68; IC: 1.48-4.82), use of common grass (OR=5.93; IC: 1.31-26.82), history of abortion in the last 12 months (OR=2.37; IC: 1.09-5.16) and presence of wildlife animals (OR=8.86; IC: 1.11-70.73) were identified as risk factors for BoHV-1 infection in the multivariate logistic regression. These results indicate that BoHV-1 infection is widespread in the studied region and that factors related to the herd characteristic and management are associated with the infection.

Abstract in English:

Spilki F.R., Silva A.D., Batista H.B.C.R., Oliveira A.P., Winkelmann E., Franco A.C., Porciúncula J.A. & Roehe P.M. 2005. Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine. Pesquisa Veterinária Brasileira 25(1):54-58. Instituto de Pesquisa Veterinária Desidério Finamor, Fepagro-Saúde Animal, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@ufrgs.br Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of respiratory, reproductive disease and abortion in cattle. Vaccination is widely applied to minimize losses induced by BoHV-1 infections; however, vaccination of dams during pregnancy with modified live virus (MLV) vaccines has been occasionally associated to abortions. We have previously reported the development of a BoHV-1 recombinant virus, constructed with basis on a Brazilian BoHV-1 (Franco et al. 2002a) from which the gene coding for glycoprotein E (gE) was deleted (gE-) by genetic manipulation. Such recombinant has been previously evaluated in its potential as a differential vaccine (gE- vaccine) that allows differentiation between vaccinated and infected animals. Here, in the first part of the present study, the safety of the gE- vaccine during pregnancy was evaluated by the intramuscular inoculation of 107.4 tissue culture 50 % infective doses (TCID50) of the virus into 22 pregnant dams (14 BoHV-1 seronegative; 8 seropositive), at different stages of gestation. Other 15 pregnant dams were kept as non-vaccinated controls. No abortions, stillbirths or fetal abnormalities were seen after vaccination. Seroconversion was observed in both groups of previously seronegative vaccinated animals. In the second part of the study, the potential of the gE- vaccine virus to spread among beef cattle under field conditions was examined. Four heifers were inoculated intranasally with a larger amount (107,6 TCID50) of the gE- vaccine (to increase chances of transmission) and mixed with other sixteen animals at the same age and body condition, in the same grazing area, at a population density equal to the average cattle farming density within the region (one cattle head per 10,000 m2), for 180 days. All animals were monitored daily for clinical signs. Serum samples were collected on days 0, 30, 60 and 180 post-vaccination. Seroconversion was observed only in vaccinated heifers. These results indicate that, under the conditions of the present study, the gE- vaccine virus did not cause any noticeable harmful effect on pregnant dams and on its offspring and did not spread horizontally among cattle.

• Bovine herpesvirus 1 BoHV-1 recombinant gE- vaccine.

Abstract in Portuguese:

Spilki F.R., Silva A.D., Batista H.B.C.R., Oliveira A.P., Winkelmann E., Franco A.C., Porciúncula J.A. & Roehe P.M. 2005. Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine. Pesquisa Veterinária Brasileira 25(1):54-58. Instituto de Pesquisa Veterinária Desidério Finamor, Fepagro-Saúde Animal, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@ufrgs.br Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of respiratory, reproductive disease and abortion in cattle. Vaccination is widely applied to minimize losses induced by BoHV-1 infections; however, vaccination of dams during pregnancy with modified live virus (MLV) vaccines has been occasionally associated to abortions. We have previously reported the development of a BoHV-1 recombinant virus, constructed with basis on a Brazilian BoHV-1 (Franco et al. 2002a) from which the gene coding for glycoprotein E (gE) was deleted (gE-) by genetic manipulation. Such recombinant has been previously evaluated in its potential as a differential vaccine (gE- vaccine) that allows differentiation between vaccinated and infected animals. Here, in the first part of the present study, the safety of the gE- vaccine during pregnancy was evaluated by the intramuscular inoculation of 107.4 tissue culture 50 % infective doses (TCID50) of the virus into 22 pregnant dams (14 BoHV-1 seronegative; 8 seropositive), at different stages of gestation. Other 15 pregnant dams were kept as non-vaccinated controls. No abortions, stillbirths or fetal abnormalities were seen after vaccination. Seroconversion was observed in both groups of previously seronegative vaccinated animals. In the second part of the study, the potential of the gE- vaccine virus to spread among beef cattle under field conditions was examined. Four heifers were inoculated intranasally with a larger amount (107,6 TCID50) of the gE- vaccine (to increase chances of transmission) and mixed with other sixteen animals at the same age and body condition, in the same grazing area, at a population density equal to the average cattle farming density within the region (one cattle head per 10,000 m2), for 180 days. All animals were monitored daily for clinical signs. Serum samples were collected on days 0, 30, 60 and 180 post-vaccination. Seroconversion was observed only in vaccinated heifers. These results indicate that, under the conditions of the present study, the gE- vaccine virus did not cause any noticeable harmful effect on pregnant dams and on its offspring and did not spread horizontally among cattle.