Serological response against bovine herpesvirus and bovine viral diarrhea virus induced by commercial vaccines in Holstein heifers / Resposta sorológica contra herpesvírus bovino e vírus da diarreia viral bovina induzida por vacinas comerciais em novilhas Holandesas

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%)...

A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%)...(AU)

Serological response against bovine herpesvirus and bovine viral diarrhea virus induced by commercial vaccines in Holstein heifers / Resposta sorológica contra herpesvírus bovino e vírus da diarreia viral bovina induzida por vacinas comerciais em novilhas Holandesas

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus.(AU)

A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.(AU)

Serological response against bovine herpesvirus and bovine viral diarrhea virus induced by commercial vaccines in Holstein heifers

ABSTRACT: Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus.

RESUMO: A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.

Potencial antiviral e virucida da melitina e apamina contra herpesvírus bovino tipo 1 e vírus da diarreia viral bovina / Antiviral and virucidal potential of melittin and apamin against bovine herpesvirus type 1 and bovine viral diarrhea virus

A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 μg/ml e apamina não demonstrou toxicidade à maior concentração testada (100μg/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2μg/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5μg/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando efeito citopático do vírus. Na associação das duas substâncias houve queda no título de BVDV e observou-se maior viabilidade celular quando comparados à ação isolada dos composto sobre este vírus. Isso se confirma na atividade virucida, uma vez que houve decréscimo de 90% das partículas virais de BVDV com a associação dos dois compostos do veneno de abelhas. Atuando individualmente, melitina apresentou efeito antiviral e virucida frente ao BoHV-1, zerando seu título em apenas 2 horas a 37oC. Conclui-se que melitina tem ação antiviral e virucida frente ao BoHV-1 e sua associação com apamina potencializou seus efeitos frente ao BVDV.(AU)

The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100μg/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2μg/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of bovine herpesvirus type-1. In lower concentrations (1 and 1.5μg/mL) melittin no antiviral activity because cell viability was low, showing cytopathic effect of the virus. At the association two substances there were a decrease in the title of BVDV and there was higher cell viability when compared to the isolated action of the compounds of this virus. This is confirmed in the virucidal activity, since there was a decrease of 90% of the viral particles of BVDV with the combination of the two compounds of bee venom. Acting individually, melittin showed antiviral effect and virucidal against for BoHV-1, zeroing its title in just 2 hours at 37°C. It is concluded that melittin has antiviral and virucidal action against the BoHV-1 and its association with apamin potentiate its effects against BVDV.(AU)

Potencial antiviral e virucida da melitina e apamina contra herpesvírus bovino tipo 1 e vírus da diarreia viral bovina / Antiviral and virucidal potential of melittin and apamin against bovine herpesvirus type 1 and bovine viral diarrhea virus

A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 μg/ml e apamina não demonstrou toxicidade à maior concentração testada (100μg/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2μg/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5μg/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando...(AU)

The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100μg/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2μg/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of...(AU)

Potencial antiviral e virucida da melitina e apamina contra herpesvírus bovino tipo 1 e vírus da diarreia viral bovina

ABSTRACT: The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100g/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2g/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of bovine herpesvirus type-1. In lower concentrations (1 and 1.5g/mL) melittin no antiviral activity because cell viability was low, showing cytopathic effect of the virus. At the association two substances there were a decrease in the title of BVDV and there was higher cell viability when compared to the isolated action of the compounds of this virus. This is confirmed in the virucidal activity, since there was a decrease of 90% of the viral particles of BVDV with the combination of the two compounds of bee venom. Acting individually, melittin showed antiviral effect and virucidal against for BoHV-1, zeroing its title in just 2 hours at 37°C. It is concluded that melittin has antiviral and virucidal action against the BoHV-1 and its association with apamin potentiate its effects against BVDV.

RESUMO: A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 g/ml e apamina não demonstrou toxicidade à maior concentração testada (100g/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2g/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5g/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando efeito citopático do vírus. Na associação das duas substâncias houve queda no título de BVDV e observou-se maior viabilidade celular quando comparados à ação isolada dos composto sobre este vírus. Isso se confirma na atividade virucida, uma vez que houve decréscimo de 90% das partículas virais de BVDV com a associação dos dois compostos do veneno de abelhas. Atuando individualmente, melitina apresentou efeito antiviral e virucida frente ao BoHV-1, zerando seu título em apenas 2 horas a 37oC. Conclui-se que melitina tem ação antiviral e virucida frente ao BoHV-1 e sua associação com apamina potencializou seus efeitos frente ao BVDV.

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV)

ABSTRACT: Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.

RESUMO: Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57g/mL), quando comparado aos extratos verde (0,78g/mL) e marrom (0,39g/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.

Immune response in holstein calves vaccinated at six months of age

Background: Calves are agammaglobulinemic and immature at birth and their immunological defense must be improved by colostrum, although, maternal antibodies decrease after two months of age and calves are susceptible to Bovine Respiratory Disease Complex (BRDC). Then, this research evaluated the immune response to BVDV and BoHV-1 in young calves to prevent BRDC.Materials, Methods & Results: Ten male Holstein calves were distributed in two groups with five animals on each: nonvaccinated VAC (-) and vaccinated VAC (+). Calves were vaccinated twice at 180 and 210 days of age. It was selected a commercial multivalent vaccine containing inactivated isolated of BVDV type-1 (strains 5960) and BVDV type-2 (strains 53637), and modified-live BoHV-1 (strains RLB103), with Quil A, cholesterol and Amphigen as adjuvant. The immune response (IR) was evaluated at 180 (T0), 210 (T1) and 240 (T2) days of life by serum neutralization (SN) and immunophenotyping. Specific antibodies to BVDV were detected in 40% (2/5) and 60% (3/5) of vaccinated calves at T1 and T2, respectively. Specific antibodies (Abs) to BoHV-1 were observed in 40% (2/5) at T1 and 100% (5/5) at T2 in the VAC (+) group. Titers of Abs to BoHV-1 increase from T0 to T2 (P = 0.034) in VAC (+). Moreover, geometric mean titer (GMT) to BoHV-1 in VAC (+) group was higher than VAC (-) after secondary IR in T2 (P = 0.006). The proportion of the markers T lymphocytes subpopulation (CD3+ , CD4+ , CD8+ and WC1+ ) and B lymphocytes (CD21+ ) were similar in the two experimental groups, while the expression of the CD25+ marker by gamma-delta (WC1+ ) was higher in VAC (+) at T1.Discussion: Low titers of circulating Abs could be observed just for BoHV-1 in the calves from VAC (-) at T0, probably due to remaining Abs from dams transferred by colostrum intake. This fact indicates the need of appropriate vaccination schedules to prevent the virosis in dairy heifers.[...](AU)

Immune response in holstein calves vaccinated at six months of age

Background: Calves are agammaglobulinemic and immature at birth and their immunological defense must be improved by colostrum, although, maternal antibodies decrease after two months of age and calves are susceptible to Bovine Respiratory Disease Complex (BRDC). Then, this research evaluated the immune response to BVDV and BoHV-1 in young calves to prevent BRDC.Materials, Methods & Results: Ten male Holstein calves were distributed in two groups with five animals on each: nonvaccinated VAC (-) and vaccinated VAC (+). Calves were vaccinated twice at 180 and 210 days of age. It was selected a commercial multivalent vaccine containing inactivated isolated of BVDV type-1 (strains 5960) and BVDV type-2 (strains 53637), and modified-live BoHV-1 (strains RLB103), with Quil A, cholesterol and Amphigen as adjuvant. The immune response (IR) was evaluated at 180 (T0), 210 (T1) and 240 (T2) days of life by serum neutralization (SN) and immunophenotyping. Specific antibodies to BVDV were detected in 40% (2/5) and 60% (3/5) of vaccinated calves at T1 and T2, respectively. Specific antibodies (Abs) to BoHV-1 were observed in 40% (2/5) at T1 and 100% (5/5) at T2 in the VAC (+) group. Titers of Abs to BoHV-1 increase from T0 to T2 (P = 0.034) in VAC (+). Moreover, geometric mean titer (GMT) to BoHV-1 in VAC (+) group was higher than VAC (-) after secondary IR in T2 (P = 0.006). The proportion of the markers T lymphocytes subpopulation (CD3+ , CD4+ , CD8+ and WC1+ ) and B lymphocytes (CD21+ ) were similar in the two experimental groups, while the expression of the CD25+ marker by gamma-delta (WC1+ ) was higher in VAC (+) at T1.Discussion: Low titers of circulating Abs could be observed just for BoHV-1 in the calves from VAC (-) at T0, probably due to remaining Abs from dams transferred by colostrum intake. This fact indicates the need of appropriate vaccination schedules to prevent the virosis in dairy heifers.[...]

Analysis of isotype-specific antibody responses to bovine herpesviruses 1.1 and 1.2a allows to estimate the stage of infection

Specific IgM, IgA, IgG1, IgG2, as well as neutralizing antibody responses were evaluated in sera of calves experimentally infected with two isolates of bovine herpesvirus type 1 (BoHV1) of distinct subtypes (subtype 1, BoHV1.1; subtype 2a, BoHV-1.2a). No significant differences were observed in the antibody responses induced by each BoHV-1 subtype. The antibody responses following primary acute infection were characterized by an increase in specific IgM and IgA levels between days 2 and 14 post inoculation (pi). IgG1 was detected from days 11 to 30 pi. IgG2 was detected on the sample taken on day 30 pi. Reactivation of infection following dexamethasone administration induced a significant rise in IgA levels, whereas IgG1 and IgG2 levels, which were at high levels from the beginning of the reactivation process, showed a slight alteration after corticosteroid treatment. These results suggest that it is possible to estimate the dynamics of BoHV-1 infections with basis on the analysis of class- and subclass-specific antibody responses. Such information may be particularly useful for the study of the kinetics of the infection in a herd and to aid in the adoption of appropriate control measures..

In vitro evaluation of the antiviral potential of Guettarda angelica against animal herpesviruses / Avaliação in vitro do potencial antiviral de Guettarda angelica contra herpesvírus animais

Background: The number of antiviral studies using plant extracts has increased in the last decades, and the results have shown that plants are potential sources of compounds that are able to inhibit and/or decrease viral infections. The selection of these plants by ethnopharmacological criteria increases the probability of fi nding new substances with signifi cant pharmacological and biological activities. Hence, Brazil has an advantage in this area due to its extensive biodiversity and ethnological diversity. Guettarda angelica is a plant from the Brazilian Caatinga region the roots of which are popularly used for various therapeutic purposes, including veterinary use. The aim of this work was to investigate the in vitro antiviral activity of extracts of plant parts from G. angelica against three animal herpesviruses: bovine (BoHV-1), suid (SuHV-1) and equine (EHV-1) herpesviruses type 1.Materials, Methods & Results: The extracts of roots, leaves and seeds of G. angelica were initially screened for in vitro antiviral activity against these herpesviruses using the virus yield reduction assay. The MDBK cells were used in assays with BoHV-1 and SuHV-1, and the Vero cells with EHV-1. For these assays, the cells previously treated with the extracts in non-cytotoxic concentrations were inoculated with logarithmic dilutions of each virus. The viral inhibitory activity of extracts

Background: The number of antiviral studies using plant extracts has increased in the last decades, and the results have shown that plants are potential sources of compounds that are able to inhibit and/or decrease viral infections. The selection of these plants by ethnopharmacological criteria increases the probability of fi nding new substances with signifi cant pharmacological and biological activities. Hence, Brazil has an advantage in this area due to its extensive biodiversity and ethnological diversity. Guettarda angelica is a plant from the Brazilian Caatinga region the roots of which are popularly used for various therapeutic purposes, including veterinary use. The aim of this work was to investigate the in vitro antiviral activity of extracts of plant parts from G. angelica against three animal herpesviruses: bovine (BoHV-1), suid (SuHV-1) and equine (EHV-1) herpesviruses type 1.Materials, Methods & Results: The extracts of roots, leaves and seeds of G. angelica were initially screened for in vitro antiviral activity against these herpesviruses using the virus yield reduction assay. The MDBK cells were used in assays with BoHV-1 and SuHV-1, and the Vero cells with EHV-1. For these assays, the cells previously treated with the extracts in non-cytotoxic concentrations were inoculated with logarithmic dilutions of each virus. The viral inhibitory activity of extracts

Avaliação in vitro do potencial antiviral de Guettarda angelica contra herpesvírus animais / In vitro evaluation of the antiviral potential of Guettarda angelica against animal herpesviruses

Background: The number of antiviral studies using plant extracts has increased in the last decades, and the results have shown that plants are potential sources of compounds that are able to inhibit and/or decrease viral infections. The selection of these plants by ethnopharmacological criteria increases the probability of fi nding new substances with significant pharmacological and biological activities. Hence, Brazil has an advantage in this area due to its extensive biodiversity and ethnological diversity. Guettarda angelica is a plant from the Brazilian Caatinga region the roots of which are popularly used for various therapeutic purposes, including veterinary use. The aim of this work was to investigate the in vitro antiviral activity of extracts of plant parts from G. angelica against three animal herpesviruses: bovine (BoHV-1), suid (SuHV-1) and equine (EHV-1) herpesviruses type 1. Materials, Methods & Results: The extracts of roots, leaves and seeds of G. angelica were initially screened for in vitro antiviral activity against these herpesviruses using the virus yield reduction assay. The MDBK cells were used in assays with BoHV-1 and SuHV-1, and the Vero cells with EHV-1. For these assays, the cells previously treated with the extracts in non-cytotoxic concentrations were inoculated with logarithmic dilutions of each virus. The viral inhibitory activity of extracts was calculated by difference of virus titer between treated infected cells and non-treated infected cells. Only the aqueous extract from seeds (AEs) showed a significant antiviral activity (P < 0.01, ANOVA followed by Tukey test) against all herpesviruses leading continuous studies. Thus, the selectivity index (SI) of this extract was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay by calculating the ratio CC50 over IC50. The 50% cytotoxic concentration (CC50) was defined as the extract concentration that reduced the cell viability by 50% when compared to untreated controls; the 50% inhibitory concentration (IC50) was defined as the concentration of the extract that inhibited 50% of viral replication when compared to the virus control. The CC50 and IC50 were obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 5 Demo program and represented the means ± standard deviation of three independent experiments. The CC50 for Vero cells was 400.60 ± 0.20 µg/mL, while the CC50 for MDBK cells was 920.50 ± 0.19 µg/mL. The IC50 values of the AEs on the BoHV-1, SuHV-1 and EHV-1 were 22.79 µg/mL, 91.30 µg/mL and 19.95 µg/mL, respectively. The SI values of this extract for each virus obtained from these data were 40.39, 10.08 and 20.08 for BoHV-1, SuHV-1, and EHV-1, respectively. Discussion: To ensure the antiviral activity of a plant extract and consequently its future use as antiviral agent is crucial the obtainment of its selectivity index or safety index. It is guarantee of a true antiviral effect and not the result of cytotoxicity of the extract on cells, and that could be confused with an antiviral activity. Other important point are the extract IC50 values less than 100 µg/mL. The results of the AEs of G. angelica are in accordance with these considerations indicating that the G. angelica seeds may be a potential source of antiviral compounds insurance and encouraging further investigation of them.

Prevalence of bovine viral diarrhoea, bovine herpesvirus type 1 and 4 infections in repeat breeding cows in Western Turkey / Prevalência de infecções por vírus da diarreia viral bovina e herpesvírus tipos 1 e 4 em fêmeas bovinas repetidoras do cio na região oeste da Turquia

Bovine Viral Diarrhoea Virus, Infectious Bovine Rhinotracheitis and Bovine Herpesvirus Type 4 infections are widelyknown viral infections as the reproductive system pathogen in cattle. The purpose of this study was to investigate thepresence and prevalence of these infections in Repeat Breeding diagnosed cows. A total of 139 blood samples werecollected from nine different dairy herds in Western Turkey. All sampled animals were clinically healthy but not becomepregnant even though they were three to eleven times artificially inseminated. The samples were controlled serologicallyfor BHV1 and BHV4 using ELISA test, 2% and 3.9% proportions were detected, respectively. These values were foundto be very low in comparison to other studies in Turkey. Out of 139, 81 (58.2%) cows were found to be seropositive forBVDV, and the percentage was above the 70% in six herds. BVD antigen was detected in two animals in a herd with71.4% antibody rates. Antigen positive animals were sampled and tested again four weeks later and persistent viremiawere confirmed. In conclusion, BVDV infection may be responsible for repeat breeding in studied herds.(AU)

Infecções por Vírus da Diarréia Viral Bovina (BVDV), Vírus da Rinotraqueíte Infecciosa Bovina (BHV-1) e HerpesvírusTipo 4 (BHV-4) são amplamente reconhecidas como infecções do trato reprodutivo em bovinos. O objetivo desteestudo foi investigar a presença e a prevalência destas infecções em fêmeas bovinas repetidoras de cio. Um total de 139amostras de sangue foi colhido de nove diferentes propriedades leiteiras na região Oeste da Turquia. Todos os animaisamostrados estavam clinicamente saudáveis, embora não tenham sido enxertados por inseminação artificial após trêsa onze tentativas. As amostras de soro foram testadas por método ELISA para detecção de anticorpos anti-BHV-1 eBHV-4, e 2,0 e 3,9% das amostras, respectivamente, foram consideradas positivas. Estes valores foram consideradosbaixos quando comparados com os de outros estudos na Turquia. De 139 amostras, 81 (58,2%) foram positivas para apresença de anticorpos anti-BVDV e mais de 70% das propriedades foram reveladas positivas. O antígeno de BVDVfoi detectado em dois animais em uma propriedade em que 71,4% dos animais eram soropositivos. Animais positivospara a presença de antígenos foram amostrados e testados novamente quatro semanas depois e viremia persistente foidetectada. Concluindo, infecção por BVDV pode ser responsável pelas perdas reprodutivas nas propriedades estudadas.(AU)

Anticorpos contra o herpesvírus bovino tipo 1 (BoHV-1) nas regiões de planejamento do estado de Goiás, Brasil / Antibodies against bovine herpevirus type 1 (BoHV-1) in the ten planning regions og Goiás state, Brazil

Este estudo investigou a presença e o título de anticorpos contra o herpesvírus bovino tipo 1 (BoHV-1) em bovinos em idade de abate pertencentes às dez regiões de planejamento do Estado de Goiás. Para tanto, 660 amostras de soro foram colhidas em frigoríficos sob inspeção federal e submetidas à pesquisa de anticorpos neutralizantes pelo teste de vírus-neutralização. Os resultados mostraram que 84,5% das amostras de soro foram reagentes para o BoHV-1, ao passo que as análises regionalizadas detectaram índices que variaram de 63,4% a 100%. Evidenciaram-se 36,3% de animais com elevados títulos de anticorpos neutralizantes. Nesse sentido, a Região do Entorno do Distrito Federal apresentou a maior porcentagem de animais com títulos elevados (65,8%) e a Região do Sudoeste Goiano, a menor (13,6%). A ocorrência de grande número de bovinos reagentes com elevados títulos de anticorpos sugere o curso de infecções ativas ou reativadas nesses animais.

This study investigated the presence of antibodies and antibodies titers against bovine herpesvirus type 1 (BoHV-1) in bovines at slaughter age in the ten planning regions of Goiás State. Blood samples (660) were collected in slaughterhouses under federal inspection and they were tested for neutralizing antibody by virus neutralization test. The results showed that 84.5% of serum samples were positive for BoHV-1, while the regionalized analysis found rates varying from 63.4% to 100%. This study revealed 36.3% of animals with high titers of neutralizing antibodies. The region surrounding Brasilia presented the highest percentage of animals with high titers (65.8%) and the Southeast Region of Goiás, the lowest (13.6%). The occurrence of high percentage of reagents with high antibody levels suggests the course of BoHV-1 active or reactivated infections among these animals.

Anticorpos contra o herpesvírus bovino tipo 1 (BoHV-1) nas regiões de planejamento do estado de Goiás, Brasil / Antibodies against bovine herpevirus type 1 (BoHV-1) in the ten planning regions og Goiás state, Brazil

Este estudo investigou a presença e o título de anticorpos contra o herpesvírus bovino tipo 1 (BoHV-1) em bovinos em idade de abate pertencentes às dez regiões de planejamento do Estado de Goiás. Para tanto, 660 amostras de soro foram colhidas em frigoríficos sob inspeção federal e submetidas à pesquisa de anticorpos neutralizantes pelo teste de vírus-neutralização. Os resultados mostraram que 84,5% das amostras de soro foram reagentes para o BoHV-1, ao passo que as análises regionalizadas detectaram índices que variaram de 63,4% a 100%. Evidenciaram-se 36,3% de animais com elevados títulos de anticorpos neutralizantes. Nesse sentido, a Região do Entorno do Distrito Federal apresentou a maior porcentagem de animais com títulos elevados (65,8%) e a Região do Sudoeste Goiano, a menor (13,6%). A ocorrência de grande número de bovinos reagentes com elevados títulos de anticorpos sugere o curso de infecções ativas ou reativadas nesses animais.(AU)

This study investigated the presence of antibodies and antibodies titers against bovine herpesvirus type 1 (BoHV-1) in bovines at slaughter age in the ten planning regions of Goiás State. Blood samples (660) were collected in slaughterhouses under federal inspection and they were tested for neutralizing antibody by virus neutralization test. The results showed that 84.5% of serum samples were positive for BoHV-1, while the regionalized analysis found rates varying from 63.4% to 100%. This study revealed 36.3% of animals with high titers of neutralizing antibodies. The region surrounding Brasilia presented the highest percentage of animals with high titers (65.8%) and the Southeast Region of Goiás, the lowest (13.6%). The occurrence of high percentage of reagents with high antibody levels suggests the course of BoHV-1 active or reactivated infections among these animals.(AU)

Herpesvírus bovinos (BoHV-1.1 e BoHV-1.2b) em forma infecciosa em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul / Bovine herpesviruses (BoHV-1 and BoHV-1.2b) in infectious form in brains of cattle submitted to rabies diagnosis in the State of Rio Grande do Sul, Brazil

Verificou-se a incidência de herpesvírus bovinos (BoHVs) em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul. Para tanto, amostras coletadas durante dois anos (n=70) foram submetidas ao isolamento viral em cultivos celulares. Os BoHVs foram isolados em dois (2,9 por cento) encéfalos. Após serem submetidas à caracterização antigênica e molecular, as amostras foram subtipadas como BoHV-1.1 e BoHV-1.2b. A BoHV-1.1 foi isolada de um encéfalo que foi também positivo para raiva. O vírus da raiva foi identificado em 11 amostras (15,7 por cento). Estes achados revelam que a incidência de BoHVs em forma infecciosa em bovinos com encefalite foi baixa, embora represente 16,7 por cento (2/12) dos encéfalos nos quais um agente viral foi identificado. Tal fato confirma a já reportada associação entre BoHV-1 e encefalites. Esse é o primeiro relato da ocorrência de BoHV-1.2b, um subtipo considerado menos patogênico, em um caso de doença neurológica em bovinos.(AU)

The incidence of bovine herpesviruses (BoHVs) was determined in brains of cattle submitted to rabies diagnosis in the State of Rio Grande do Sul, Brazil. Brain samples collected in a two-year interval (n=70) were submitted to virus isolation in cell culture. The BoHVs were isolated from two (2.9 percent) of the brains. After the antigenic and molecular characterization, the isolated strains were subtyped as BoHV-1.1 and BoHV-1.2b. The BoHV-1.1 isolate was recovered from a brain sample that was also positive for rabies. Rabies virus was identified in 11 (15.7 percent) samples. These findings reveal that the incidence of infectious BoHVs in brains of cattle with encephalitis was low, although these represented 16.7 percent (2/12) of samples from which at least one viral agent was identified. This confirms the previously reported link between BoHV-1 and bovine encephalitis. However, it is the first report on the association of BoHV-1.2b, a putatively less pathogenic BoHV subtype, with neurological disease in cattle.(AU)

Influência dos anticorpos maternos na resposta imune induzida pela vacinação em bezerros Holandeses

O objetivo geral desta pesquisa foi avaliar a transferência de imunidade passiva e a sua influência na resposta vacinal para as viroses envolvidas na Doença Respiratória Bovina (DRB). Os dados obtidos nesta pesquisa estão apresentados em dois capítulos. Capítulo 1 - Objetivou-se avaliar a dinâmica de anticorpos (Acs) específicos para as viroses respiratórias e subpopulações de linfócitos em bezerros do nascimento aos 240 dias (d) de vida. Para tanto, acompanhou-se a transferência de imunidade passiva de Acs específicos para as viroses respiratórias em 19 bezerros, destes cinco foram selecionados para acompanhamento da dinâmica de Acs neutralizantes e subpopulações de linfócitos dos 14 aos 240d. O colostro fornecido era proveniente de vacas doadoras vacinadas. A análise da qualidade individual do colostro revelou índice Brix 21%, observando-se forte correlação desses valores com proteína total (r= 0,942 e P= 0,001). Após 48 horas da ingestão do colostro, pôde-se observar soroconversão dos 19 animais (100%) para os agentes virais envolvidos na DBR. As medianas (Log2) encontradas foram de 12,3, 9,0, 5,0 e 8,5 para BVDV, BoHV-1, BRSV e BPIV-3. Dos 14 aos 240d os 5 bezerros avaliados, demonstraram declínio gradual dos títulos de Acs (Log2) para BVDV (12,8-3,3), BoHV-1 (10,0-3,3) e BPIV-3 (10,0-2,0), embora o BVDV não tenha apresentado soronegatividade até os 240d. Manifestações clínicas de broncopneumonia foram observadas em 4/5 (80%) bezerros dos 80 aos 135 d. BRSV (11,3-2,0) apresentou perfil diferenciado na dinâmica de anticorpos em relação às demais viroses. Não foram detectadas diferenças estatísticas entre os momentos para as viroses apesar das variações detectadas (P>0,05). A meia-vida e tempo para soronegatividade foram de 36,2±6,1 e 367,01±68,7d para BVDV, 50,7±18,0 e 239,67±66,88d para BoHV-1 e, 46,8±21,1 e 303,36±60,15d para BPIV-3. BRSV não respeitou modelo de regressão para cálculos de meia-vida e soronegatividade. Valores absolutos e relativos das populações de linfócitos não revelaram diferenças estatísticas entre os momentos (P>0,05). Contudo, a dinâmica das subpopulações de linfócitos revelou aumento de células B CD21+ até 150d; aumento nos valores relativos das subpopulações CD4+, CD8+ e CD3+CD4-CD8- principalmente aos 74-90d. Assim, pôde-se determinar uma janela de susceptibilidade a partir dos 74d especialmente ao BRSV e BoHV-1, momento que precede o aumento dos títulos de Acs após exposição natural. Capítulo 2 Objetivou-se avaliar a influência dos Acs maternos na resposta imune para DRB induzida pela vacinação. Foram selecionados 23 bezerros recém-nascidos, distribuídos aleatoriamente entre 4 grupos experimentais: G1 vacinado aos 14d e booster aos 44d; G2 aos 90d e aos 120d; G3 aos 180d e aos 210d. Além disso manteve-se um grupo controle não vacinado CG1, CG2 e CG3. Os bezerros foram vacinados com a mesma vacina comercial empregada para vacinação das doadoras de colostro. Observou-se: (1) a vacina com BVDV inativado não promoveu aumento dos títulos de Acs para nenhum dos grupos avaliados; (2) G1 não demonstrou soroconversão para nenhuma das viroses, enquanto controle CG1 exibiu decréscimo dos títulos para BVDV, BoHV-1 e BPIV-3; (3) o BRSV apresentou baixa soroconversão no G2, enquanto o controle demonstrou altos títulos dos 44-120d; (4) não foi possível distinguir entre quais tempos ocorreram diferenças entre títulos (P>0,0167); (5) linfócitos B (CD21+) aumentaram do T0-T2 para G1, diminuíram para G2, e aumentaram no G3; (6) linfócitos T CD3+ diminuíram ao longo do tempo para todos os grupos, exceto CG3; (7) apesar de oscilações, linfócitos T CD4+, CD8+ e WC1+ se mantiveram praticamente constantes até 240d, exibindo maiores proporções nos grupos vacinados; (8) a expressão do marcador CD25+ foi mantida pelo grupo G1 até o T2, mas apresentou aumento no G2 e G3; (9) manifestações de broncopneumonias foram identificadas nos bezerros do grupo controle (4/5 - 80%) e podem ter exercido influência nas diferenças encontradas para as células entre os grupos. Em geral, a vacinação dos bezerros aos 90 (G2) e 180d (G3) manteve ou estimulou a produção de Acs para o BoHV-1, BRSV e BPIV-3, e a ativação das células T expressas pelo marcador CD25+ pode ter sido responsável pela proteção dos bezerros frente à DRB. Assim, com base nos resultados, concluiu-se que a intensidade da imunidade dos bezerros induzida pela vacinação aumentou de acordo com o desenvolvimento etário e diminuição dos títulos de Acs maternos.

The main purpose of this research was to evaluate the transfer of passive immunity and its influence on vaccine response to the viruses involved in bovine respiratory disease (BRD). The data obtained in this study are presented in two chapters. Chapter 1 - The objective was to evaluate the dynamics of specific antibodies to the respiratory viruses and lymphocyte subpopulations in the calves from birth to 240 days (d) of life. Thus, the transfer of passive immunity of specific antibodies to the respiratory viruses were assessed in 19 calves; from these, five were selected for monitoring the dynamic of neutralizing Abs and lymphocytes subpopulations from 14 to 240d. The colostrum was provided from donor vaccinated cows. The analysis of individual quality of the colostrum revealed Brix 21%, observing strong correlation of these values with total protein (r = 0.942 and P = 0.001). After 48 hours of colostrum intake, was observed seroconversion of the 19 animals (100%) for the viral agents involved in the DBR. The median (Log2) ratio found was 12.3, 9.0, 5.0 and 8.5 for BVDV, BoHV-1, BRSV and BPIV-3. The 5 calves followed from 14 to 240d showed gradual decline in antibody titers (Log2) to BVDV (12.8 to 3.3), BoHV-1 (10.0 to 3.3) and BPIV-3 (10, 0-2.0), although could not be detected seronegative calves for BVDV up to eight months of age. Clinical manifestations of bronchopneumonia were observed in 4/5 (80%) calves from 80 to 135 days of life. BRSV (11.3 to 2.0) showed a distinct profile in the dynamics of antibodies compared to other viruses. There were no statistical differences between times for viruses despite variations detected (P> 0.05). The half-life and time to become seronegative were 36.2±6.1 and 367.01±68.7d for BVDV, 50.7±18.0 and 239.67±66.88d for BoHV-1 and, 46.8±21.1 and 303.36±60.15d for BPIV-3. BRSV did not respect regression model to perform half-life and seronegative calculations. Absolute and relative values of lymphocyte populations revealed no statistical differences between times (P> 0.05). However, the dynamics of lymphocyte subpopulations showed increase in B cells CD21+ up to 150d; increase in the relative values of CD4+, CD8+ and CD3+CD4-CD8- subpopulations, mainly to 74-90d. Thus, it was possible to determine a window of susceptibility since 74d especially for BRSV and BoHV-1, moment that precede the increase in antibody titers after natural exposure. Chapter 2 - The objective was to evaluate the influence of maternal antibodies in the immune response to respiratory viruses induced by vaccination. Was selected 23 newborn calves that were randomly distributed in four groups: G1 - vaccinated at 14d and booster at 44d; G2 - vaccinated at 90d and booster at 120d; G3 - vaccinated at 180d and booster at 210d. Furthermore were kept a non-vaccinated control group - CG1, CG2 and CG3. Calves were vaccinated with the same commercial vaccine in the colostrum donors. From these, could be observed: (1) the vaccine with inactivated BVDV did not promote increase of antibodies titers in any of the assessed groups; (2) G1 did not demonstrated seroconversion for any of the viruses while CG1 control exhibited decrease in the titers for BVDV, BoHV-1 and BPIV-3; (3) BRSV had low seroconversion in G2, while the control showed high titers from 44 to 120d; (4) where the differences between times occurred could not be distinguished (P <0.0167); (5) B cells (CD21+) increased from T0 to T2 for G1, decreased for G2, and increased for G3; (6) T lymphocytes CD3+ decreased over time for all groups except for CG3; (7) despite variations, T lymphocytes CD4+, CD8+ and WC1+ remained almost constant until 240d, displaying greater proportions in the vaccinated groups; (8) the expression of the CD25+ marker was maintained in the vaccinated group G1 up to T2, whereas vaccination promoted an increase of this expression in G2 and G3; (9) clinical manifestations of bronchopneumonia were identified in the control group (4/5 calves - 80%) and may have influence on the differences found for the cells between the groups. In general, vaccination of calves at 90 (G2) and 180d (G3) maintained or stimulated the production of Abs to BoHV-1, BRSV and BPIV-3, and the activation of T cells expressed by the CD25+ marker may have been responsible for the protection of calves from BRD. Thus, based on the results, it was concluded that the intensity of the immunity induced by vaccination of calves increased according to the age of development and decay of maternal antibody titers

Replicação e excreção viral durante a infecção aguda e após a reativação da latência induzida por dexametasona em bezerros inoculados com os herpesvírus bovinos tipo 1 (BHV-1) e 5 (BHV-5)

The efficiency of the establishment and reactivation of latent infection by bovine herpesviruses types 1 and 5 (BHV-1 and 5) was compared. Calves inoculated intranasally with BHV-1 (SV-265, n=6) or BHV-5 (SV-507, n=6) presented a mild to moderate nasal discharge and shed virus in nasal secretions in titers up to 10(7.81)TCID50/ml (mean tissue culture infectious dose) during an average of 10.5 days (6-15 [BHV-1]) or up to 10(6.7) TCID50/ml during 15.3 days (13-18 [BHV-5]). Dexamethasone administration (Dx; 0.5mg/kg) at day 60pi resulted in reactivation of the infection in all calves. Virus shedding in nasal secretions was detected in titers up to 10(5.5)TCID50/ml during 6 to 9 days (mean: 7.8) in calves inoculated with BHV-1 and in titers up to 10(6.1)TCID50/ml (3 to 12 days, mean: 7.5) in calves inoculated with BHV-5. These results showed that BHV-1 and BHV-5 were capable of establishing and reactivating the latent infection at comparable levels.

Nesse estudo, comparou-se a eficiência de estabelecimento e reativação da infecção latente pelos herpesvírus bovinos tipos 1 e 5 (BHV-1 e 5) em bezerros. Bezerros inoculados pela via intranasal com uma amostra de BHV-1 (SV-265, n=6) ou BHV-5 (SV-507, n=6) apresentaram corrimento nasal discreto a moderado e excretaram vírus em secreções nasais em títulos de até 10(7,81)DICC50/ml (dose infectante para 50% dos cultivos celulares) durante um período médio de 10,5 dias (6-15 [BHV-1]) ou títulos de até 10(6,7) DICC50/ml durante 15,3 dias (13-18 [BHV-5]). A administração de dexametasona (Dx; 0,5mg/kg, via endovenosa) aos 60 dias pós-inoculação (pi) resultou em reativação da infecção em todos os animais inoculados. O vírus foi detectado em secreções nasais dos bezerros inoculados com o BHV-1 em títulos de até 10(5,5)DICC50/ml por 6 a 9 dias (x:7,8) e em títulos de até 10(6,1)DICC50/ml (durante 3 a 12 dias, x: 7,5 dias) nos bezerros inoculados com o BHV-5. Os resultados obtidos demonstraram que tanto o BHV-1 quanto o BHV-5 foram capazes de estabelecer e reativar a infecção latente em níveis semelhantes.