PESQUISA VETERINÁRIA BRASILEIRA

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.- Brum M.C.S., Caron L., Chowdhury S.I., Weiblen R. & Flores E.F. 2010. Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E. Pesquisa Veterinária Brasileira 30(1):57-62. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com The immunogenicity of an inactivated, experimental vaccine based on a bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E (BoHV-5 gE/TKD) was evaluated in cattle and the results were compared with a vaccine containing the parental BoHV-5 strain (SV507/99). To formulate the vaccines, each virus (wildtype SV507/99 and BoHV-5 gE/TKD) was multiplied in cell culture and inactivated with binary ethyleneimine (BEI). Each vaccine dose contained approximately of 107.5 TCID50 of inactivated virus mixed with an oil-based adjuvant (46:54). Forty calves, 6 to 9-months-old, were allocated into two groups of 20 animals each and vaccinated twice (days 0 and 22pv) by the subcutaneous route with either vaccine. Serum samples collected at day 0 and at different intervals after vaccination were tested for virus neutralizing (VN) antibodies against the parental virus and against heterologous BoHV-5 and BoHV-1 isolates. The VN assays demonstrated seroconversion to the respective homologous viruses in all vaccinated animals after the second vaccine dose (mean titers of 17.5 for the wildtype vaccine; 24.1 for the recombinant virus). All animals remained reagents up to day 116 pv, yet showing a gradual reduction in VN titers. Animals from both vaccine groups reacted in similar VN titers to different BoHV-1 and BoHV-5 isolates, yet the magnitude of serological response of both groups was higher against BoHV-5 field isolates. Calves vaccinated with the recombinant virus did not develop antibodies to gE as verified by negative results in a gE-specific ELISA, what would allow serological differentiation from naturally infected animals. Taken together, these results indicate that inactivated antigens of BoHV-5 gE/TK recombinant virus induced an adequate serological response against BoHV-5 and BoHV-1 and thus can be used as an alternative, differential vaccine candidate.

• Herpesvirus type 5 thymidine kinase glycoprotein E

Abstract in Portuguese:

RESUMO.- Brum M.C.S., Caron L., Chowdhury S.I., Weiblen R. & Flores E.F. 2010. Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E. [Imunogenicidade de uma cepa inativada do herpesvírus bovino tipo 5 defectiva na timidina quinase e glicoproteína E.] Pesquisa Veterinária Brasileira 30(1):57-62. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com A imunogenicidade de vacina experimental inativada, produzida com uma cepa do herpesvírus bovino tipo 5 defectiva nos genes da timidina quinase e glicoproteína E (BoHV-5 gE/TKD) foi avaliada em bovinos e o resultado foi comparado com a resposta induzida pela cepa parental do BoHV-5 (SV507/99). Para a formulação da vacina, cultivos de células infectados com cada um dos vírus (SV507/99 ou BoHV-5 gE/TKD) foram inativados com etilenamina binária. Cada dose de vacina continha aproximadamente 107,5 TCID50 de um dos vírus inativados emulsionado em adjuvante oleoso (46:54). Quarenta bezerros de raças cruzadas, com idade entre seis a nove meses, foram alocados em dois grupos de 20 animais cada e vacinados duas vezes (dia 0 e 22 pv) pela via subcutânea com uma das vacinas. Amostras de soro foram coletadas no dia 0 e a vários intervalos após vacinação para a pesquisa de anticorpos neutralizantes frente ao vírus homólogo ou frente a isolados de BoHV-5 e BoHV-1. Os testes de soroneutralização (SN) demonstraram que todos os animais soroconverteram após a segunda dose da vacina (títulos médios de 17,5 para o grupo SV507/99; 24,1 para o grupo BoHV-5 gE/TKD). Todos os animais mantiveram níveis de anticorpos neutralizantes até o dia 116 pv, no entanto foi observada uma redução gradual no títulos. A sorologia cruzada com amostras heterólogas do BoHV-5 e BoHV-1 indicou que ambos os grupos vacinais reagiram em níveis similares frente ao mesmo vírus, no entanto a magnitude da resposta sorológica foi maior frente a amostras de BoHV-5. Os animais vacinados com a cepa recombinante não desenvolveram anticorpos contra a gE detectáveis por um ELISA específico, o que permitiria a sua diferenciação sorológica de animais infectados naturalmente. Esses resultados demonstram que a vacina contendo antígenos inativados do vírus recombinante BoHV-5 gE/TKD induziu resposta sorológica em níveis satisfatórios, constituindo-se, assim, em alternativa a cepa vacinal diferencial.

Abstract in English:

ABSTRACT.- Arenhart S., Bauermann F.V., Oliveira S.A.M., Weiblen R. & Flores E.F. 2009. [Shedding and transmission of bovine viral diarrhea virus by persistently infected calves.] Excreção e transmissão do vírus da diarréia viral bovina por bezerros persistentemente infectados. Pesquisa Veterinária Brasileira 29(9):736-742. Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: eduardofurtadoflores@gmail.com Persistently infected (PI) calves born to cows infected with non-cytopathic bovine virus diarrhea virus (BVDV) represent the main reservoir of the virus in nature. We herein report an investigation on the patterns of virus shedding and transmission by five PI calves produced experimentally through inoculation of pregnant cows with Brazilian BVDV isolates. Five calves that survived intrauterine infection were born healthy, lacking neutralizing antibodies to BVDV and harboring virus in the blood. After weaning - and following the disappearance of colostral antibodies - PI calves were monitored for virus in serum and body secretions (ocular, oral, nasal and genital) at weekly intervals for up to 150 days. For each animal, the virus titers in serum showed minor variations throughout the collections (with one exception that presented an increase late in infection), yet the titers varied widely among animals (from 102 to 106TCID50/mL). Virus shedding in secretions was detected steadily during all the observation period with minor titer variations for each particular animal. The highest titers were generally detected in nasal and ocular secretions (titers 104 to 106TCID50mL) whereas genital and oral secretions usually contained low amount of virus (102 to 103TCID50mL). To evaluate the kinetics of virus transmission by these animals, one PI was introduced on a group of 10 seronegative calves maintained with a high animal density simulating the conditions of an intensive management. All 10 contact calves seroconverted to BVDV by day 30. Another PI calf was introduced into a 48-head herd kept under a low animal density, extensive grass management. Among these animals, 8/48 (16.6%) seroconverted by day 10, 26/48 (54.1%) by day 40 and 37/48 (77%) were seropositive at day 100, when the monitoring was discontinued. These results show that continuous viremia and virus shedding in high titers in secretions by PI animals assure an efficient and rapid virus transmission to contact animals, being the kinetics of transmission much faster under intensive conditions.

• Bovine viral diarrhea virus BVDV vírua da diarréia viral bovina

Abstract in Portuguese:

RESUMO.- Arenhart S., Bauermann F.V., Oliveira S.A.M., Weiblen R. & Flores E.F. 2009. [Shedding and transmission of bovine viral diarrhea virus by persistently infected calves.] Excreção e transmissão do vírus da diarréia viral bovina por bezerros persistentemente infectados. Pesquisa Veterinária Brasileira 29(9):736-742. Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: eduardofurtadoflores@gmail.com Bezerros persistentemente infectados (PI) nascidos de vacas infectadas com amostras não-citopáticas do vírus da diarréia viral bovina (BVDV) se constituem nos principais reservatórios do vírus na natureza. Este trabalho relata uma investigação do padrão de excreção e transmissão viral por cinco bezerros PI produzidos experimentalmente pela inoculação de vacas prenhes com isolados brasileiros do BVDV. Cinco bezerros que sobreviveram a infecção intrauterina nasceram saudáveis, soronegativos e com a presença de vírus no sangue. Após o desmame - e desaparecimento dos anticorpos colostrais - os bezerros PI foram monitorados semanalmente durante 150 dias para a presença de vírus e títulos virais no soro e em secreções (ocular, oral, nasal e genital). Os títulos virais no soro de cada animal apresentaram pequenas variações durante o período (com exceção de um animal que apresentou um aumento de título tardiamente), mas os títulos variaram amplamente entre os animais (entre 102 e 106TCID50/ml). O vírus também foi excretado continuamente nas secreções de todos os animais, com pequenas variações de título entre as coletas. Os maiores títulos virais foram geralmente detectados nas secreções nasais e oculares (títulos de 104 a 106TCID50/mL), enquanto as secreções orais e genitais usualmente continham títulos virais baixos (102 a 103TCID50/mL). Com o objetivo de avaliar a dinâmica de transmissão viral, um bezerro PI foi introduzido em um grupo de 10 bezerros soronegativos, mantido com uma alta densidade animal e submetido a manejo diário para simular as condições de manejo semi-intensivo. Após 30 dias de convívio com o bezerro PI, todos os demais animais haviam soroconvertido ao BVDV. Para investigar a transmissão viral sob condições extensivas, outro bezerro PI foi incorporado a um rebanho de 48 animais mantido a campo, com baixa densidade animal e submetido a manejo extensivo. Dentre estes animais, 8/48 (16,6%) foram soropositivos para anticorpos no dia 10, 26/48 (54,1%) no dia 40 e 37/48 (77%) haviam soroconvertido no dia 100, quando encerrou-se o monitoramento. Estes resultados demonstram que a viremia e excreção viral contínua em altos títulos por animais PI assegura a transmissão rápida do BVDV a animais mantidos em contato, sendo a transmissão notadamente mais rápida em condições intensivas e de alta densidade animal.

Abstract in English:

Abstract.- Oliveira E.C., Sonne L., Bezerra Júnior P.S., Teixeira E.M., Dezengrini R., Pavarini S.P., Flores E.F. & Driemeier D. 2009. [Clinic and pathological findings in dogs naturally infected with canine herpesvirus.] Achados clínicos e patológicos em cães infectados naturalmente por herpesvírus canino. Pesquisa Veterinária Brasileira 29(8):637-642. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br The clinic and pathological findings and laboratorial testing of puppies with postmortem diagnosis of canine herpesvirus infection are described. The cases occurred in two households of Porto Alegre, RS, in April 2007 and July 2008. The puppies presented anorexia, depression, cry and dyspnea, followed by death about 24-72 hours after the onset of clinical signs. At necropsy multifocal pin point hemorrhages were observed in the kidneys. The liver was enlarged with petechiae and white foci on the surface. The lungs were red and did not collapse. The spleen was enlarged and, in some cases, with petechiae on the capsular surface. Mesenteric lymph nodes and thymus were enlarged. Microscopic lesions included hemorrhages and multifocal necrosis of the renal tubular epithelial cells, hepatocytes and lymphoid tissues. In the lungs there was severe multifocal alveolar necrosis with abundant fibrin deposits and a mixed inflammatory infiltrate of variable intensity. Intranuclear inclusion bodies were identified in the periphery of necrotic areas in hepatocytes, renal tubular epithelial cells and alveolar cells. Samples of liver, kidney and lung were positive in the direct immunofluorescence test for canine herpes virus type 1 (CHV-1). The diagnosis was based on epidemiological data, necropsy findings, histological lesions and positive immunofluorescence results using CHV-1 antibody in tissue samples. To our knowledge, this is the first report of the identification of CHV-1 in Brazil, although previous clinic and pathological findings already suggested the presence of the virus in Brazilian canine population.

• Canine herpesvirus

Abstract in Portuguese:

RESUMO.- Oliveira E.C., Sonne L., Bezerra Júnior P.S., Teixeira E.M., Dezengrini R., Pavarini S.P., Flores E.F. & Driemeier D. 2009. [Clinic and pathological findings in dogs naturally infected with canine herpesvirus.] Achados clínicos e patológicos em cães infectados naturalmente por herpesvírus canino. Pesquisa Veterinária Brasileira 29(8):637-642. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Descrevem-se os achados clínicos e patológicos e os exames laboratoriais de filhotes de cães com diagnóstico post mortem de infecção por herpesvírus canino. Os casos ocorreram em duas propriedades da Cidade de Porto Alegre, Rio Grande do Sul, em abril de 2007 e julho de 2008. Clinicamente, os cães apresentaram anorexia, apatia, choro e dispneia. A morte dos cães ocorreu após 24-72 horas do início dos sinais clínicos. Na necropsia observaram-se hemorragia multifocal renal e hepatomegalia com petéquias e pontos brancos na superfície natural do fígado. Os pulmões se apresentaram não-colapsados e vermelhos. Havia esplenomegalia e, em alguns cães, petéquias na superfície capsular do baço. Aumento dos linfonodos mesentéricos e do timo foi observado. Lesões microscópicas incluíram hemorragia e necrose multifocal em células epiteliais tubulares renais, hepatócitos e tecidos linfoides. Nos pulmões, havia necrose alveolar multifocal acentuada com abundante material fibrinoso e infiltrado inflamatório misto de intensidade variada. Ocasionais corpúsculos de inclusão intranucleares em áreas periféricas à necrose foram identificados em hepatócitos, células epiteliais de túbulos renais e células alveolares. Amostras de fígado, rim e pulmão foram positivas na imunofluorescência direta para herpesvírus canino tipo 1 (CHV-1). O diagnóstico de infecção por herpesvírus foi baseado nos achados de necropsia, histológicos e de imunofluorescência positiva em tecidos usando anticorpo anti-CHV-1. De nosso conhecimento, este é o primeiro relato da identificação do CHV-1 no Brasil, embora achados clínico-patológicos anteriores já sugerissem a presença do agente na população canina do país.

Abstract in English:

ABSTRACT.- Flores E.F., Weiblen R, Vogel F.S.F., Dezengrini R., Almeida S.R., Spilki F.R. & Roehe P.M. 2009. [Experimental neuropathogenesis of bovine herpesvirus 5 infection in rabbits.] Neuropatogênese experimental da infecção pelo herpesvírus bovino tipo 5 em coelhos. Pesquisa Veterinária Brasileira 29(1):1-16. Departamento de Medicina Veterinária Preventiva. Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS. Brazil. E-mail: eduardofurtadoflores@gmail.com Several aspects of the biology of bovine herpesvirus 5 (BoHV-5) have been studied in rabbits, which develop acute infection and neurological disease upon experimental inoculation. The acute infection is followed by the establishment of latent infection, which can be naturally or artificially reactivated. The first experiments in rabbits established a protocol for virus inoculation and monitoring the infection, and characterized the main virological, clinical and pathological aspects of the acute infection. The pathogenesis of acute infection, from the initial viral replication at site of inoculation, pathways and kinetics of viral transport to the brain, distribution and virus replication in the central nervous system (CNS), cellular and tissue tropism, clinical signs and CNS pathology have been extensively studied using this animal model. Subsequently, several biological and molecular aspects of latent BoHV-5 infection have also been elucidated upon inoculation of rabbits. Rabbits have also been used to investigate the phenotype (neuroinvasiveness, neurogrowth) of field isolates and recombinant vaccine candidates, protection by passive immunity, vaccine protection, the efficacy of anti-viral drugs and support therapies for neurological disease. This animal model was also used to investigate the origin and distribution of electric impulses involved in seizures - a hallmark of BoHV-5 induced neurological infection - and also to test the efficacy of anti-convulsivants. In spite of the possible differences between rabbits and cattle - the natural host of the virus - the observations taken from this experimental model have greatly contributed to the knowledge of the biology of BoHV-5 infection. The present article presents a review of the main published and unpublished results and observations by our group, comprising more than a decade of studies on the pathogenesis of BoHV-5 infection in the rabbit model.

• Bovine herpesvirus 5 BoHV-5 rabbits experimental model

Abstract in Portuguese:

ABSTRACT.- Flores E.F., Weiblen R, Vogel F.S.F., Dezengrini R., Almeida S.R., Spilki F.R. & Roehe P.M. 2009. [Experimental neuropathogenesis of bovine herpesvirus 5 infection in rabbits.] Neuropatogênese experimental da infecção pelo herpesvírus bovino tipo 5 em coelhos. Pesquisa Veterinária Brasileira 29(1):1-16. Departamento de Medicina Veterinária Preventiva. Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS. Brazil. E-mail: eduardofurtadoflores@gmail.com Several aspects of the biology of bovine herpesvirus 5 (BoHV-5) have been studied in rabbits, which develop acute infection and neurological disease upon experimental inoculation. The acute infection is followed by the establishment of latent infection, which can be naturally or artificially reactivated. The first experiments in rabbits established a protocol for virus inoculation and monitoring the infection, and characterized the main virological, clinical and pathological aspects of the acute infection. The pathogenesis of acute infection, from the initial viral replication at site of inoculation, pathways and kinetics of viral transport to the brain, distribution and virus replication in the central nervous system (CNS), cellular and tissue tropism, clinical signs and CNS pathology have been extensively studied using this animal model. Subsequently, several biological and molecular aspects of latent BoHV-5 infection have also been elucidated upon inoculation of rabbits. Rabbits have also been used to investigate the phenotype (neuroinvasiveness, neurogrowth) of field isolates and recombinant vaccine candidates, protection by passive immunity, vaccine protection, the efficacy of anti-viral drugs and support therapies for neurological disease. This animal model was also used to investigate the origin and distribution of electric impulses involved in seizures - a hallmark of BoHV-5 induced neurological infection - and also to test the efficacy of anti-convulsivants. In spite of the possible differences between rabbits and cattle - the natural host of the virus - the observations taken from this experimental model have greatly contributed to the knowledge of the biology of BoHV-5 infection. The present article presents a review of the main published and unpublished results and observations by our group, comprising more than a decade of studies on the pathogenesis of BoHV-5 infection in the rabbit model.

Abstract in English:

ABSTRACT.- Arenhart S., Silva L.F., Henzel A., Ferreira R., Weiblen R. & Flores E.F. 2008. [Fetal protection against bovine viral diarrhea virus (BVDV) in pregnant cows previously immunized with an experimental attenuated vaccine.] Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada. Pesquisa Veterinária Brasileira 28(10):461-470. Departamento de Medicina Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br This paper reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of bovine viral diarrhea virus (BVDV-1 and BVDV-2). Cows (n=19) were vaccinated twice, with a 34-days-interval, with the experimental vaccine and together with non-vaccinated controls (n=18), were mated and challenged between days 60 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1,280- ³10,240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1,280). At the end of the monitoring, only three out of 18 control cows (16.6%) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3%) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8%) delivered virus-positive calves; five were healthy and survived (27.7%); two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4%) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4%) vaccinated cows delivery virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet this fetus could not be examined for the presence of virus. Another cow delivered a virus-positive calf (5.2%). In summary, the experimental vaccine induced adequate antibody titers in most animals and the immunological response induced by vaccination was able to prevent fetal infection and reproductive losses upon challenge with a pool of heterologous BVDV isolates. Hence, this experimental vaccine may be an attractive alternative for the prevention of reproductive losses associated with BVDV infection.

• Bovine viral diarrhea virus BVDV experimental vaccine fetal protection

Abstract in Portuguese:

ABSTRACT.- Arenhart S., Silva L.F., Henzel A., Ferreira R., Weiblen R. & Flores E.F. 2008. [Fetal protection against bovine viral diarrhea virus (BVDV) in pregnant cows previously immunized with an experimental attenuated vaccine.] Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada. Pesquisa Veterinária Brasileira 28(10):461-470. Departamento de Medicina Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br This paper reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of bovine viral diarrhea virus (BVDV-1 and BVDV-2). Cows (n=19) were vaccinated twice, with a 34-days-interval, with the experimental vaccine and together with non-vaccinated controls (n=18), were mated and challenged between days 60 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1,280- ³10,240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1,280). At the end of the monitoring, only three out of 18 control cows (16.6%) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3%) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8%) delivered virus-positive calves; five were healthy and survived (27.7%); two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4%) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4%) vaccinated cows delivery virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet this fetus could not be examined for the presence of virus. Another cow delivered a virus-positive calf (5.2%). In summary, the experimental vaccine induced adequate antibody titers in most animals and the immunological response induced by vaccination was able to prevent fetal infection and reproductive losses upon challenge with a pool of heterologous BVDV isolates. Hence, this experimental vaccine may be an attractive alternative for the prevention of reproductive losses associated with BVDV infection.

Abstract in English:

ABSTRACT.- Ferreira L.C.L., Flores E.F., Driemeier D., Melo O. & Lemos R.A.A. 2008. [Mucosal disease associated with generalized dermatitis in cattle, Mato Grosso do Sul, Brazil.] Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul. Pesquisa Veterinária Brasileira 28(6):285-292. Departamento de Medicina Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Av. Senador Filinto Müller 2443, Campo Grande, MS 79070-900, Brazil. E-mail: eqrural@nin.ufms.br This paper reports epidemiological, clinical, pathological and laboratory diagnostic aspects of a form of dermatitis associated with mucosal disease (MD) in cattle. It also focuses on the methods used for identifying persistently infected (PI) animals and on the impact of the disease on fertility and weaning rates in the affected herd. Cases of dermatitis associated with MD were diagnosed in two 12 and 24-month-old Nelore calves belonging to a beef cattle farm that operates the full cycle of production (calving, rearing, finishing) in Mato Grosso do Sul, Midwest Brazil. The clinical signs exhibited by affected cattle 0included slow, progressive weight loss; formation of diffuse skin crusts in multiple body areas; skin dryness; multiple ulcerations on the gums and dorsal surface of the tongue which evolved to longitudinal fissures; formation of keratinized projections; and detachment of hoof horn. In addition, diarrhea affected one animal in the late stage of the disease. Necropsies also revealed longitudinal erosions in the esophagus. Histological examination showed coagulation necrosis foci in esophageal and lingual mucosae, with neutrophil and lymphocyte infiltration. Skin lesions consisted of epidermal coagulation necrosis associated with neutrophil infiltration and hyperkeratosis. In both cases, clinical diagnosis was confirmed by the isolation and identification of cytopathic and noncytopathic biotypes of the bovine viral diarrhea virus (BVDV) and immunohistochemical detection of viral antigens in formalin fixed tissues. Out of 300 cattle that had contact with the affected animals, 38 were found to be seropositive - in high neutralizing titers - to BVDV. Blood samples from 1,025 young animals and 40 bulls from the farm were examined for the presence of BVDV to identify potential PI animals. The virus was isolated from blood of three calves in the initial test and, 12 months later, from two of them which had remained on the farm. Only one of these calves was found to be BVDV-positive by immuno-histochemical testing performed on ear-tissue samples. In the year following the birth of PI calves, the herd underwent decreases in fertility and weaning rates, which later returned to their previous levels. The resulting data demonstrate the presence of BVDV infection in beef herds in Mato Grosso do Sul and provide evidence as to include the disease in the differential diagnosis of causes of generalized dermatitis in cattle.

• Bovine viral diarrhea virus mucosal disease cattle diseases viral diseases dermatitis

Abstract in Portuguese:

ABSTRACT.- Ferreira L.C.L., Flores E.F., Driemeier D., Melo O. & Lemos R.A.A. 2008. [Mucosal disease associated with generalized dermatitis in cattle, Mato Grosso do Sul, Brazil.] Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul. Pesquisa Veterinária Brasileira 28(6):285-292. Departamento de Medicina Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Av. Senador Filinto Müller 2443, Campo Grande, MS 79070-900, Brazil. E-mail: eqrural@nin.ufms.br This paper reports epidemiological, clinical, pathological and laboratory diagnostic aspects of a form of dermatitis associated with mucosal disease (MD) in cattle. It also focuses on the methods used for identifying persistently infected (PI) animals and on the impact of the disease on fertility and weaning rates in the affected herd. Cases of dermatitis associated with MD were diagnosed in two 12 and 24-month-old Nelore calves belonging to a beef cattle farm that operates the full cycle of production (calving, rearing, finishing) in Mato Grosso do Sul, Midwest Brazil. The clinical signs exhibited by affected cattle 0included slow, progressive weight loss; formation of diffuse skin crusts in multiple body areas; skin dryness; multiple ulcerations on the gums and dorsal surface of the tongue which evolved to longitudinal fissures; formation of keratinized projections; and detachment of hoof horn. In addition, diarrhea affected one animal in the late stage of the disease. Necropsies also revealed longitudinal erosions in the esophagus. Histological examination showed coagulation necrosis foci in esophageal and lingual mucosae, with neutrophil and lymphocyte infiltration. Skin lesions consisted of epidermal coagulation necrosis associated with neutrophil infiltration and hyperkeratosis. In both cases, clinical diagnosis was confirmed by the isolation and identification of cytopathic and noncytopathic biotypes of the bovine viral diarrhea virus (BVDV) and immunohistochemical detection of viral antigens in formalin fixed tissues. Out of 300 cattle that had contact with the affected animals, 38 were found to be seropositive - in high neutralizing titers - to BVDV. Blood samples from 1,025 young animals and 40 bulls from the farm were examined for the presence of BVDV to identify potential PI animals. The virus was isolated from blood of three calves in the initial test and, 12 months later, from two of them which had remained on the farm. Only one of these calves was found to be BVDV-positive by immuno-histochemical testing performed on ear-tissue samples. In the year following the birth of PI calves, the herd underwent decreases in fertility and weaning rates, which later returned to their previous levels. The resulting data demonstrate the presence of BVDV infection in beef herds in Mato Grosso do Sul and provide evidence as to include the disease in the differential diagnosis of causes of generalized dermatitis in cattle.

Abstract in English:

ABSTRACT.- Henzel A., Diel D.G., Arenhart S., Vogel F.S.F., Weiblen R. & Flores E.F. 2008. [Virological and clinico-pathological features of acute vulvovaginitis and latent infection by bovine herpesvirus 1.2 in heifers experimentally infected.] Aspectos virológicos e clínico-patológicos da infecção genital aguda e latente pelo herpesvírus bovino tipo 1.2 em bezerras experimentalmente infectadas. Pesquisa Veterinária Brasileira 28(3):140-148. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Venereal infection of heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) may result in vulvovaginitis and transient infertility. The acute infection is followed by the establishment of latent infection which can be periodically reactivated. We herein describe the virology and clinico-pathological aspects of acute and recrudescent vulvovaginitis in heifers inoculated with a Brazilian BoHV-1.2 isolate recovered from an outbreak of balanoposthitis. Genital inoculation of isolate SV-56/90 (108.1TCID50/animal) in four eight-months-old heifers resulted in efficient virus replication in the genital mucosa and the development of moderate to severe vulvovaginitis. The inoculated heifers shed virus in genital secretions in titers up to 107.3TCID50/mL until day 10 pi and developed genital congestion, swelling, vesicles and pustules. The vesicles and pustules increased in size eventually coalesced and became covered with a yellowish exsudate. These signs appeared at day 2 pi, increased in severity up to days 5 - 8 pi and progressively subsided thereafter. Dexamethasone administration at day 55 pi resulted in virus shedding in vaginal secretions for up to 10 days. Virus reactivation in all animals was accompanied by clinical recrudescence of the disease, yet less severe than during acute infection. Examination of sacral ganglia and lymph nodes by PCR at day 36 post-reactivation revealed the presence of latent viral DNA in the pudendal (4/4), genito-femoral, sciatic and rectal caudal (3/4) and obturator nerve ganglia (1/4); in addition to several regional lymph nodes. These results demonstrate the virulence of isolate SV-56/90 for heifers and pave the way for its use in further pathogenesis studies and vaccine-challenge trials.

• Bovine herpesvirus BoHV-1.2 vulvovaginitis genital infection latency reactivation

Abstract in Portuguese:

ABSTRACT.- Henzel A., Diel D.G., Arenhart S., Vogel F.S.F., Weiblen R. & Flores E.F. 2008. [Virological and clinico-pathological features of acute vulvovaginitis and latent infection by bovine herpesvirus 1.2 in heifers experimentally infected.] Aspectos virológicos e clínico-patológicos da infecção genital aguda e latente pelo herpesvírus bovino tipo 1.2 em bezerras experimentalmente infectadas. Pesquisa Veterinária Brasileira 28(3):140-148. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Venereal infection of heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) may result in vulvovaginitis and transient infertility. The acute infection is followed by the establishment of latent infection which can be periodically reactivated. We herein describe the virology and clinico-pathological aspects of acute and recrudescent vulvovaginitis in heifers inoculated with a Brazilian BoHV-1.2 isolate recovered from an outbreak of balanoposthitis. Genital inoculation of isolate SV-56/90 (108.1TCID50/animal) in four eight-months-old heifers resulted in efficient virus replication in the genital mucosa and the development of moderate to severe vulvovaginitis. The inoculated heifers shed virus in genital secretions in titers up to 107.3TCID50/mL until day 10 pi and developed genital congestion, swelling, vesicles and pustules. The vesicles and pustules increased in size eventually coalesced and became covered with a yellowish exsudate. These signs appeared at day 2 pi, increased in severity up to days 5 - 8 pi and progressively subsided thereafter. Dexamethasone administration at day 55 pi resulted in virus shedding in vaginal secretions for up to 10 days. Virus reactivation in all animals was accompanied by clinical recrudescence of the disease, yet less severe than during acute infection. Examination of sacral ganglia and lymph nodes by PCR at day 36 post-reactivation revealed the presence of latent viral DNA in the pudendal (4/4), genito-femoral, sciatic and rectal caudal (3/4) and obturator nerve ganglia (1/4); in addition to several regional lymph nodes. These results demonstrate the virulence of isolate SV-56/90 for heifers and pave the way for its use in further pathogenesis studies and vaccine-challenge trials.

Abstract in English:

ABSTRACT.- Almeida S.R., Diel D.G., Rissi D.R., Weiblen R. & Flores E.F. 2008. [Clinic and pathological characterization of acute mammillitis in lactating ewes inoculated with bovine herpesvirus 2.] Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes inoculadas experimentalmente com o herpesvírus bovino 2. Pesquisa Veterinária Brasileira 28(1):87-94. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Mammillitis caused by bovine herpesvirus type 2 (BoHV-2) is an important disease in dairy herds yet its pathogenesis remains largely unknown. This report describes the reproduction and characterization of acute mammillitis in lactating ewes inoculated with BoHV-2 in the skin of the udder and teats. Five out of eight inoculated ewes developed large plaques, with focal necrosis, small vesicles and crust formation in the inoculated areas. The lesions were first observed on day 4 post-inoculation (pi), progressed in size and severity up to days 7-8pi and subsided progressively thereafter. Infectious virus was isolated from the lesions at days 7 and 8pi. Viral antigens and herpesvirus-like particles were demonstrated by electron microscopy in lesions examined at days 5, 6 and 10pi. Histological findings included epithelial necrosis, erosions and ulcers, and formation of syncytial cells. Intranuclear inclusions bodies in epithelial, syncytial and inflammatory cells and lymphoplasmacytic inflammatory infiltrate in the dermis were also observed. In a second experiment, seven out of ten lambs inoculated into the nostrils and muzzle developed nasal hyperemia and discharge, vesicles, and erosions in the nose. Infectious virus was isolated from lesions during up to three days and all lambs seroconverted to BoHV-2. Attempts to reactivate the latent infection by dexamethasone administration on day 40pi failed, since virus shedding, clinical recrudescence or seroconversion were not observed. The reproduction of acute infection and mammillitis resembling that occurring in cattle paves the way for the use of sheep to study several aspects of the biology of BoHV-2 infection.

• Bovine herpesvirus-2 BoHV-2 mammillitis sheep experimental infection

Abstract in Portuguese:

ABSTRACT.- Almeida S.R., Diel D.G., Rissi D.R., Weiblen R. & Flores E.F. 2008. [Clinic and pathological characterization of acute mammillitis in lactating ewes inoculated with bovine herpesvirus 2.] Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes inoculadas experimentalmente com o herpesvírus bovino 2. Pesquisa Veterinária Brasileira 28(1):87-94. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Mammillitis caused by bovine herpesvirus type 2 (BoHV-2) is an important disease in dairy herds yet its pathogenesis remains largely unknown. This report describes the reproduction and characterization of acute mammillitis in lactating ewes inoculated with BoHV-2 in the skin of the udder and teats. Five out of eight inoculated ewes developed large plaques, with focal necrosis, small vesicles and crust formation in the inoculated areas. The lesions were first observed on day 4 post-inoculation (pi), progressed in size and severity up to days 7-8pi and subsided progressively thereafter. Infectious virus was isolated from the lesions at days 7 and 8pi. Viral antigens and herpesvirus-like particles were demonstrated by electron microscopy in lesions examined at days 5, 6 and 10pi. Histological findings included epithelial necrosis, erosions and ulcers, and formation of syncytial cells. Intranuclear inclusions bodies in epithelial, syncytial and inflammatory cells and lymphoplasmacytic inflammatory infiltrate in the dermis were also observed. In a second experiment, seven out of ten lambs inoculated into the nostrils and muzzle developed nasal hyperemia and discharge, vesicles, and erosions in the nose. Infectious virus was isolated from lesions during up to three days and all lambs seroconverted to BoHV-2. Attempts to reactivate the latent infection by dexamethasone administration on day 40pi failed, since virus shedding, clinical recrudescence or seroconversion were not observed. The reproduction of acute infection and mammillitis resembling that occurring in cattle paves the way for the use of sheep to study several aspects of the biology of BoHV-2 infection.

Abstract in English:

ABSTRACT.- Silva M.S., Brum M.C.S., Weiblen R. & Flores E.F. 2007. [Identification and differentiation of herpesvirus types 1 and 5 isolated from clinical samples in central-southern Brazil, Argentina and Uruguay (1987-2006).] Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006). Pesquisa Veterinária Brasileira 27(10):403-408. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine herpesviruses types 1 and 5 (BoHV-1; BoHV-5) are genetically and antigenically closely related such they can not be distinguished by routine diagnostic tests. As BoHV-1 has been historically associated with respiratory and genital disease, herpesviruses isolated from these clinical syndromes have been tentatively – and sometimes definitively - diagnosed as BoHV-1. Likewise, cases of herpetic neurological infection in cattle have been generally attributed to BoHV-5. This study reports the identification of 40 herpesvirus isolates from different clinical specimens and syndromes in central-southern Brazil, Argentina and Uruguay (1987-2006) by the use of a PCR able to differentiate between BoHV-1 and BoHV-5. BoHV-1 isolates (n=16) were identified in cases of respiratory disease (n=3), vulvovaginitis and/or balanoposthitis (n=3), in semen of healthy bulls (n=5) and in cases of neurological disease (n=5). Viruses identified as BoHV-5 (n=24) were isolated predominantly from cases of neurological disease (n=21), but also from semen of healthy bulls (n=2) and from a spleen of a calf with systemic disease (n=1). These results show that both BoHV-1 and BoHV-5 are not strictly associated with their respective diseases; yet are frequently involved in clinical conditions otherwise attributed to the other virus. These findings also reinforce the need of correctly identifying the herpesvirus isolates as to better understand their pathogenesis and epidemiology.

• Bovine herpesvirus BoHV-1 BoHV-5 differential diagnosis epidemiology

Abstract in Portuguese:

ABSTRACT.- Silva M.S., Brum M.C.S., Weiblen R. & Flores E.F. 2007. [Identification and differentiation of herpesvirus types 1 and 5 isolated from clinical samples in central-southern Brazil, Argentina and Uruguay (1987-2006).] Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006). Pesquisa Veterinária Brasileira 27(10):403-408. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine herpesviruses types 1 and 5 (BoHV-1; BoHV-5) are genetically and antigenically closely related such they can not be distinguished by routine diagnostic tests. As BoHV-1 has been historically associated with respiratory and genital disease, herpesviruses isolated from these clinical syndromes have been tentatively – and sometimes definitively - diagnosed as BoHV-1. Likewise, cases of herpetic neurological infection in cattle have been generally attributed to BoHV-5. This study reports the identification of 40 herpesvirus isolates from different clinical specimens and syndromes in central-southern Brazil, Argentina and Uruguay (1987-2006) by the use of a PCR able to differentiate between BoHV-1 and BoHV-5. BoHV-1 isolates (n=16) were identified in cases of respiratory disease (n=3), vulvovaginitis and/or balanoposthitis (n=3), in semen of healthy bulls (n=5) and in cases of neurological disease (n=5). Viruses identified as BoHV-5 (n=24) were isolated predominantly from cases of neurological disease (n=21), but also from semen of healthy bulls (n=2) and from a spleen of a calf with systemic disease (n=1). These results show that both BoHV-1 and BoHV-5 are not strictly associated with their respective diseases; yet are frequently involved in clinical conditions otherwise attributed to the other virus. These findings also reinforce the need of correctly identifying the herpesvirus isolates as to better understand their pathogenesis and epidemiology.