Natural association between bovine leukemia virus and reproductive infectious diseases

Background: Enzootic bovine leukosis (EBL) is a widespread infectious disease caused by the bovine leukemia virus (BLV), which results in immune system dysfunction. The resulting immunosuppression may lead to an increased prevalence of other diseases. Dairy cows infected have altered immune function associated with decreased milk production and shortened lifespan and decreased immune response to immunization. BLV infection, however, is often asymptomatic, so any connection between subclinical infection and common reproductive diseases remains unknown. This study aimed to describe the relationship between naturally occurring subclinical BLV and infectious reproductive diseases seroconversion in the field. Materials, Methods & Results: The diseases investigated included Bovine viral diarrhoea (BVD), Bovine alfaherpesvirus 1 (BoHV-1), Bovine gammaherpesvirus 4 (BoHV-4), Chlamydiosis, Leptospirosis, Brucellosis and Neosporosis in dairy cattle. Six hundred fifty-five sera samples from the northern and south-central regions of Uruguay, from asymptomatic female Holstein and Holstein crosses without a history of vaccination against reproductive diseases were processed using reference diagnostic methods (Seronautalization, ELISA, MAT, Rose Bengal Plate test). The seroprevalence of BLV was 20.0%. Seroprevalence of reproductive diseases BVD, BoHV-1 and BoHV-4 were 99.3%, 41.2% and 27.3% of the populations, respectively, and the total seroprevalence of Leptospirosis, bovine Neospora caninum and Chlamydiosis were 19.8%, 29.8% and 33.0% respectively. The results revealed positive associations between naturally contracted BLV and the presence of antibodies against BoHV-1 (P = 0.002), as well as between naturally contracted BLV and presence of antibodies against Leptospira spp. (P = 0.028). Discussion: BLV infection can impact innate and adaptive immune system cells and alter the proper functioning of uninfected cells. BLV infection may also induce changes in the complex balance of cytokine expression, cell proliferation, and programmed cell death in T- and B-lymphocytes, which is critical for immune competence and effective response to infectious challenges. The progression of BLV infection has a substantial effect on host defense mechanisms. Indeed, lowmagnitude serologic responses to a commercial foot-and-mouth disease vaccine and a J5 Escherichia coli vaccine have been observed. These results are supported by recent trial studies showing a reduced immune response to vaccination against BoHV-1 and Leptospira spp. in asymptomatic animals infected with BLV. These are 2 of the most prevalent infectious reproductive diseases in cattle worldwide, and our results provide evidence that a link between BLV and susceptibility to these diseases may exist. Although there is evidence of the co-occurrence of these diseases, it remains unknown whether there is a direct or indirect effect of BLV on infertility, embryonic loss, or abortion. Another possibility is that natural infection with these reproductive pathogens (BoHV-1, Leptospira, or others) promotes BLV expression, negatively affecting the farms where these pathogens are endemic. Considering the high seroprevalence of BLV in dairy herds in North and South America where the infection is endemic, it was explored BLV's role as an immunosuppressant by quantifying its co-occurrence with diseases that affect reproductive performance in breeding herds. Future work should clarify the role of BLV and the co-occurring pathogens in causing infertility or abortions.

Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1) / Comprometimento na taxa de maturação nuclear em ovócitos de vacas naturalmente infectadas pelo herpesvírus bovino 1 (BoHV-1)

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

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

Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1) / Comprometimento na taxa de maturação nuclear em ovócitos de vacas naturalmente infectadas pelo herpesvírus bovino 1 (BoHV-1)

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

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

Use of metaphylactic protocols based on the risk to develop bovine respiratory diseases in feedlot cattle / Efeito do uso de protocolos metafiláticos segundo o risco de doença respiratória bovina em confinamentos

ABSTRACT: Bovine respiratory diseases (BRD) affect production rates negatively because it compromise health and well-being of the affected animal. The hypothesis of this study was that the use of metaphylactic protocols based on the risk to develop BRD would reduce morbidity and pulmonary lesions. For this purpose, the aims of this study were to evaluate the effect of two metaphylactic protocols on the morbidity of feedlot cattle with a known sanitary history, occurrence of pulmonary lesions at slaughter, and the possible participation of Mannheimia haemolytica, Histophilus somni, Bovine alphaherpesvirus 1 (BoHV-1) and bovine respiratory syncytial virus (BRSV) in the development of BRD. An experimental study was designed in which 3,094 adult, male, cattle, were grouped according to the risk to develop BRD: a) group without metaphylaxis (n=2,104), low-risk animals; b) metaphylaxis group with oxytetracycline (n=789), moderate-risk animals; c) metaphylaxis group with tildipirosin (n=201), high-risk animals. All cattle were immunized against pathogens associated with BRD (BoHV-1, BVDV, BRSV, PI3). The morbidity for BRD was 8.2% (253/3,094); cattle within the moderate-risk group for BRD had the lowest frequency (6.1%), followed by high-risk animals with tildipirosin metaphylaxis (6.5%) and low-risk without metaphylaxis (9.1%) (P=0.019). At the abattoir, 1.2% of lungs with lesions were found. There was a difference (P=0.036) in the frequency of pulmonary lesions between healthy animals (1.1%) and those diagnosed with BRD (2.8%). Two agents associated with BRD were identified by PCR assays in the lungs (n=37) of cattle: M. haemolytica (16.2%) and H. somni (5.4%). In addition, concomitant infections involving these pathogens were identified in the lungs of two steers.

RESUMO: As doenças respiratórias dos bovinos (DRB) afetam negativamente as taxas de produção por comprometer a saúde e o bem-estar animal. A hipótese deste estudo foi que o uso de protocolos metafiláticos com base no risco de desenvolvimento de DRB reduziria a morbidade e as lesões pulmonares ao abate. Para tanto, objetivou-se avaliar o efeito de dois protocolos metafiláticos sobre a morbidade de bovinos confinados com histórico sanitário conhecido, ocorrência de lesões pulmonares no abate e possível participação de Mannheimia haemolytica, Histophilus somni, Alphaherpesvírus bovino 1 (BoHV-1) e Vírus respiratório sincicial bovino (BRSV) na DRB. Foram avaliados 3.094 bovinos adultos, machos, agrupados de acordo com o risco de desenvolvimento de DRB: a) grupo sem metafilaxia (n=2.104), animais de baixo risco; b) grupo metafilaxia com oxitetraciclina (n=789), animais de risco moderado; c) grupo metafilaxia com tildipirosina (n=201), animais de alto risco. Todos os animais foram previamente vacinados contra os principais agentes virais causadores das DRB (BoHV-1, BVDV, BRSV e PI3). A morbidade para DRB foi de 8,2% com menor frequência em animais com risco moderado e metafilaxia com oxitetraciclina (6,1%), seguido por animais com alto risco com metafilaxia com tildipirosina (6,5%) e animais com baixo risco sem metafilaxia (9,1%) (P=0,019). No frigorífico, foram encontrados 1,2% 1,2% de animais com lesões pulmonares. Houve diferença (P=0,036) na frequência de lesões pulmonares entre animais saudáveis (1,1%) e aqueles diagnosticados com DRB (2,8%). Foram identificados dois agentes associados à DRB pela técnica de PCR nos pulmões (n=37) dos bovinos: M. haemolytica (16,2%) e H. somni (5,4%). Adicionalmente, infecções concomitantes envolvendo estes potenciais patógenos foram identificadas nos pulmões de dois animais.

Use of metaphylactic protocols based on the risk to develop bovine respiratory diseases in feedlot cattle / Efeito do uso de protocolos metafiláticos segundo o risco de doença respiratória bovina em confinamentos

ABSTRACT: Bovine respiratory diseases (BRD) affect production rates negatively because it compromise health and well-being of the affected animal. The hypothesis of this study was that the use of metaphylactic protocols based on the risk to develop BRD would reduce morbidity and pulmonary lesions. For this purpose, the aims of this study were to evaluate the effect of two metaphylactic protocols on the morbidity of feedlot cattle with a known sanitary history, occurrence of pulmonary lesions at slaughter, and the possible participation of Mannheimia haemolytica, Histophilus somni, Bovine alphaherpesvirus 1 (BoHV-1) and bovine respiratory syncytial virus (BRSV) in the development of BRD. An experimental study was designed in which 3,094 adult, male, cattle, were grouped according to the risk to develop BRD: a) group without metaphylaxis (n=2,104), low-risk animals; b) metaphylaxis group with oxytetracycline (n=789), moderate-risk animals; c) metaphylaxis group with tildipirosin (n=201), high-risk animals. All cattle were immunized against pathogens associated with BRD (BoHV-1, BVDV, BRSV, PI3). The morbidity for BRD was 8.2% (253/3,094); cattle within the moderate-risk group for BRD had the lowest frequency (6.1%), followed by high-risk animals with tildipirosin metaphylaxis (6.5%) and low-risk without metaphylaxis (9.1%) (P=0.019). At the abattoir, 1.2% of lungs with lesions were found. There was a difference (P=0.036) in the frequency of pulmonary lesions between healthy animals (1.1%) and those diagnosed with BRD (2.8%). Two agents associated with BRD were identified by PCR assays in the lungs (n=37) of cattle: M. haemolytica (16.2%) and H. somni (5.4%). In addition, concomitant infections involving these pathogens were identified in the lungs of two steers.(AU)

RESUMO: As doenças respiratórias dos bovinos (DRB) afetam negativamente as taxas de produção por comprometer a saúde e o bem-estar animal. A hipótese deste estudo foi que o uso de protocolos metafiláticos com base no risco de desenvolvimento de DRB reduziria a morbidade e as lesões pulmonares ao abate. Para tanto, objetivou-se avaliar o efeito de dois protocolos metafiláticos sobre a morbidade de bovinos confinados com histórico sanitário conhecido, ocorrência de lesões pulmonares no abate e possível participação de Mannheimia haemolytica, Histophilus somni, Alphaherpesvírus bovino 1 (BoHV-1) e Vírus respiratório sincicial bovino (BRSV) na DRB. Foram avaliados 3.094 bovinos adultos, machos, agrupados de acordo com o risco de desenvolvimento de DRB: a) grupo sem metafilaxia (n=2.104), animais de baixo risco; b) grupo metafilaxia com oxitetraciclina (n=789), animais de risco moderado; c) grupo metafilaxia com tildipirosina (n=201), animais de alto risco. Todos os animais foram previamente vacinados contra os principais agentes virais causadores das DRB (BoHV-1, BVDV, BRSV e PI3). A morbidade para DRB foi de 8,2% com menor frequência em animais com risco moderado e metafilaxia com oxitetraciclina (6,1%), seguido por animais com alto risco com metafilaxia com tildipirosina (6,5%) e animais com baixo risco sem metafilaxia (9,1%) (P=0,019). No frigorífico, foram encontrados 1,2% 1,2% de animais com lesões pulmonares. Houve diferença (P=0,036) na frequência de lesões pulmonares entre animais saudáveis (1,1%) e aqueles diagnosticados com DRB (2,8%). Foram identificados dois agentes associados à DRB pela técnica de PCR nos pulmões (n=37) dos bovinos: M. haemolytica (16,2%) e H. somni (5,4%). Adicionalmente, infecções concomitantes envolvendo estes potenciais patógenos foram identificadas nos pulmões de dois animais.(AU)

Reproductive programs for beef cattle: incorporating management and reproductive techniques for better fertility

This review summarizes strategies to increase overall pregnancy rates to TAI protocols, and potential areas for improvement in reproductive management of Nelore cows. Low BCS at calving and postpartum BCS loss negatively impacted percentage of pregnant females to TAI, with primiparous cows being the most sensitive to the effects of low BCS during the postpartum period. The current reproductive management recommended for replacement heifers (with TAI on day 0) consist of intravaginal progesterone device insertion from day -35 to -23, 0.6 mg i.m. injection of estradiol cypionate on day -23, insertion of another intravaginal progesterone device and 2.0 mg i.m. injection of estradiol benzoate on day -11, 12.5 mg i.m. injection of dinoprost tromethamine on day -4, intravaginal progesterone device removal and 0.6 mg i.m. injection of estradiol cypionate on day -2, and TAI on day 0. Supplementing corn for 41 days after the first TAI of the breeding season increased pregnancy to a second AI in primiparous cows, increased final proportion of pregnancy in primiparous cows, but decreased the final proportion of pregnancy in mature cows. In turn, supplementing melengestrol acetate or Ca salts of PUFA during the expected time of luteolysis was beneficial to pregnancy rates. Vaccinating cows against BoHV-1, BVDV, and Leptospira spp., particularly when both doses were administered before TAI, improved cow reproductive performance. Cow temperament has direct implications not only on reproductive efficiency of B. indicus females, but also on overall production efficiency in cow-calf system based on B. indicus cattle. Lastly, concentration of pregnancy-associated glycoproteins was driven by the ability of pregnancy maintenance and by sire used at TAI, whereas exploring this relationship might be interesting to improve sire fertility regarding late embryonic loss in Nelore females.

Reproductive programs for beef cattle: incorporating management and reproductive techniques for better fertility

This review summarizes strategies to increase overall pregnancy rates to TAI protocols, and potential areas for improvement in reproductive management of Nelore cows. Low BCS at calving and postpartum BCS loss negatively impacted percentage of pregnant females to TAI, with primiparous cows being the most sensitive to the effects of low BCS during the postpartum period. The current reproductive management recommended for replacement heifers (with TAI on day 0) consist of intravaginal progesterone device insertion from day -35 to -23, 0.6 mg i.m. injection of estradiol cypionate on day -23, insertion of another intravaginal progesterone device and 2.0 mg i.m. injection of estradiol benzoate on day -11, 12.5 mg i.m. injection of dinoprost tromethamine on day -4, intravaginal progesterone device removal and 0.6 mg i.m. injection of estradiol cypionate on day -2, and TAI on day 0. Supplementing corn for 41 days after the first TAI of the breeding season increased pregnancy to a second AI in primiparous cows, increased final proportion of pregnancy in primiparous cows, but decreased the final proportion of pregnancy in mature cows. In turn, supplementing melengestrol acetate or Ca salts of PUFA during the expected time of luteolysis was beneficial to pregnancy rates. Vaccinating cows against BoHV-1, BVDV, and Leptospira spp., particularly when both doses were administered before TAI, improved cow reproductive performance. Cow temperament has direct implications not only on reproductive efficiency of B. indicus females, but also on overall production efficiency in cow-calf system based on B. indicus cattle. Lastly, concentration of pregnancy-associated glycoproteins was driven by the ability of pregnancy maintenance and by sire used at TAI, whereas exploring this relationship might be interesting to improve sire fertility regarding late embryonic loss in Nelore females.(AU)

Desenvolvimento e imunogenicidade de uma vacina de aplicação intravaginal contra o alphaherpesvírus bovino tipo 5 (BoHV-5) associada a subunidade B recombinante da enterotoxina termolábil de Escherichia coli (rLTB)

O sistema imune de mucosa representa a barreira inicial frente a diversos patógenos 13 que utilizam estas superfícies como porta de entrada no organismo, como é o caso 14 dos alphaherpesvírus bovinos (BoHV). Estes vírus utilizam as mucosas, 15 principalmente nasal e genital, como ponto inicial de replicação, seguido de 16 disseminação local, viremia sistêmica e disseminação neuronal. Devido à grande 17 importância das vias mucosas na transmissão dos BoHV, torna-se evidenciado o 18 interesse no desenvolvimento de vacinas que propiciem imunidade de mucosa 19 contra estes vírus. No presente estudo, 63 fêmeas bovinas da raça Braford foram 20 divididas em sete grupos de nove animais e foram inoculadas por via intravaginal 21 com BoHV-5 inativado, associado à subunidade B recombinante da enterotoxina 22 termolábil de E. coli (rLTB) como segue: G1 (2-hidroxietilcelulose + BoHV-5 + rLTB), 23 G2 (2-hidroxietilcelulose + BoHV-5), G3 (Poloxâmero 407 + BoHV-5 + rLTB), G4 24 (Poloxâmero 407 + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Controle negativo) e G7 25 (Controle positivo). A resposta humoral local e sistêmica induzida nos animais 26 inoculados foi mensurada por meio do teste ELISA indireto (IgA e IgG) e de 27 soroneutralização, e a resposta celular por Elisa direto quantitativo (IL-2 e IFN-28 Gama). Como resultados, na mucosa vaginal, apesar do grupo G5 proporcionar os 29 maiores níveis de IgG, os grupos experimentais com polímeros (G1 e G3) 30 demonstraram-se superiores estatisticamente em relação ao grupo controle positivo 31 (G7), assim como, com relação aos níveis de IgA observados na mucosa vaginal 32 aos 30, 60 e 90 dias após a primeira inoculação. Os grupos G1 e G3 também 33 proporcionaram títulos superiores de anticorpos neutralizantes (log2) em relação aos 34 demais tratamentos (p<0,01) 90 dias pós inoculação, evidenciando a eficiência dos 35 polímeros testados como sistema de entrega do antígeno. Já na mucosa nasal, 36 houve incremento dos níveis de IgA e IgG com o uso das vacinas dos grupos G1 e 37 G3, em relação ao controle positivo, G7 (p<0,05) aos 60 e 90 dias após a primeira 38 inoculação, o que enfatiza a forte relação entre os sistemas de mucosa. A 39 inoculação das vacinas experimentais também resultou em aumento nos níveis de 40 IgA e IgG no soro dos animais, mensurado por ELISA indireto, além de 41 apresentarem títulos de anticorpos neutralizantes aos 60 e 90 dias, detectados 42 através de soroneutralização. Os maiores títulos de anticorpos neutralizantes foram 43 proporcionados pelo grupo G5 (sem polímeros e intramuscular), entretanto, os níveis 44 de IL-2 e IFN-Gama obtidos com o uso dos polímeros foram superiores aos demais 1 grupos aos 30, 60 e 90 dias após primeira inoculação. A vacina intravaginal 2 demonstrou-se capaz de incrementar os níveis de IgA e IgG e de interleucinas no 3 soro e nas mucosas nasal e vaginal dos bovinos imunizados. Estes dados 4 comprovam a capacidade da vacina de aplicação intravaginal em bovinos em 5 estimular uma resposta imune local e sistêmica, além de corroborar a atividade 6 imunoestimulante, em mucosas, da rLTB. Deste modo, esta via de administração de 7 vacinas torna-se uma alternativa interessante, principalmente quando se objetiva 8 gerar proteção local contra patógenos que utilizam as superfícies mucosas como 9 porta de entrada no organismo.

The mucosal immune system represents the initial barrier against several pathogens 13 that use these surfaces as a gateway to the organism, such as bovine 14 alphaherpesviruses (BoHV). These viruses use mucous membranes, mainly nasal 15 and genital, as the initial point of replication, followed by local dissemination, 16 systemic viremia and neuronal dissemination. Due to the great importance of the 17 mucous pathways in the transmission of BoHV, the interest in the development of 18 vaccines that provide mucosal immunity against these viruses becomes evident. In 19 the present study, 63 Braford bovine females were divided into seven groups of nine 20 animals and were inoculated intravaginally with inactivated BoHV-5, associated with 21 the recombinant B subunit of the E. coli thermolabile enterotoxin (rLTB) as follows: 22 G1 (2-hydroxyethylcellulose + BoHV-5 + rLTB), G2 (2-hydroxyethylcellulose + BoHV-23 5), G3 (Poloxamer 407 + BoHV-5 + rLTB), G4 (Poloxamer 407 + BoHV-5), G5 24 (BoHV-5 + rLTB), G6 (Negative control) and G7 (Positive control). The local and 25 systemic humoral response induced in the inoculated animals were measured using 26 the indirect ELISA test (IgA and IgG) and serum neutralization, and the cellular 27 response, direct quantitative Elisa (IL-2 and IFN-Gamma). As a result, in the vaginal 28 mucosa, despite the G5 group providing the highest levels of IgG, the experimental 29 groups with polymers (G1 and G3) showed to be statistically superior in relation to 30 the positive control group (G7), as well as, in relation to the levels of IgA observed in 31 the vaginal mucosa at 30, 60 and 90 days after the first inoculation. Groups G1 and 32 G3 also provided superior titers of neutralizing antibodies (log2) in relation to the 33 other treatments (p <0.01) 90 days after inoculation, showing the efficiency of the 34 tested polymers as an antigen delivery system. In the nasal mucosa, there was an 35 increase in IgA and IgG levels with the use of vaccines in groups G1 and G3, in 36 relation to the positive control, G7 (p<0.05) at 60 and 90 days after the first 37 inoculation, which emphasizes the strong relationship between mucosal systems. 38 The inoculation of experimental vaccines also resulted in increased levels of IgA and 39 IgG in the animals' serum, measured by indirect ELISA, in addition to presenting 40 neutralizing antibody titers at 60 and 90 days, detected through serum neutralization. 41 The highest titers of neutralizing antibodies were provided by the G5 group (without 42 polymers and intramuscular), however, the levels of IL-2 and IFN-Gamma obtained 43 with the use of polymers were higher than the other groups at 30, 60 and 90 days 44 after first inoculation. The intravaginal vaccine has been shown to increase the levels 45 of IgA and IgG and interleukins in the serum and in the nasal and vaginal mucosa of 46 immunized cattle. These data prove the capacity of the vaccine for intravaginal 1 application in cattle to stimulate a local and systemic immune response, in addition to 2 corroborating the immunostimulating activity in mucous membranes of rLTB. Thus, 3 this route of administration of vaccines becomes an interesting alternative, especially 4 when the objective is to generate local protection against pathogens that use mucous 5 surfaces as a gateway into the body.

Natural infection in ovarian structures by bovine herpesvirus 1: molecular and serological detection / Infecção natural em estruturas ovarianas pelo Herpesvírus bovino 1: detecção molecular e sorológica

In this study, the polymerase chain reaction (PCR) was used to evaluate the presence of viral DNA in ovarian tissue, in the cumulus-oocyte complex (COC), follicular liquid, and blood of animals naturally infected with bovine herpesvirus-1 (BoHV-1). The serum profile of the sampled animals was also evaluated. Samples of serum, blood, ovarian tissue, follicular liquid, and COC were collected from 147 slaughterhouse animals that were not vaccinated against BoHV-1. Contaminated or insufficient samples were disregarded. Serological tests allowed the identification of serum-positive animals with neutralizing antibodies against BoHV-1. Analysis of samples by PCR revealed the presence of viral DNA in 0.9% (1/115) of the COC samples, in 4.3% (5/117) of the ovarian tissue samples, and in 2.8% (3/108) of the blood samples. Viral DNA was not detected in any of the follicular liquid samples. In serological samples, a positivity of 83.6% (117/140) was observed for BoHV-1. All PCR-positive animals, regardless of the samples analyzed, showed positivity in the serum neutralization test for the detection of BoHV-1-specific antibodies. According to these results, a high prevalence of antibodies against BoHV-1 was detected in naturally infected animals from different herds, and the molecular tests revealed the presence of viral DNA in bovine ovarian tissue, providing evidence that this might be...(AU)

Neste trabalho foi avaliada a presença do DNA viral, por meio da Reação em Cadeia da Polimerase (PCR), no tecido ovariano, nos oócitos, líquido folicular e sangue de vacas naturalmente infectadas. Também foi avaliado o perfil sorológico dos animais amostrados. Foram coletadas amostras de soro, sangue, tecido ovariano, líquido folicular e complexo cumulus-oócitos de 147 animais abatidos em frigorífico não vacinados contra o herpesvirus bovino 1 (BoHV-1). Amostras tóxicas ou insuficientes foram descartadas. Os testes sorológicos foram realizados permitindo a identificação dos animais soropositivos para anticorpos neutralizantes contra o BoHV-1. Foram realizadas as PCRs onde foi observada a presença do DNA viral em 0,9% (1/115) dos oócitos, em 4,3% (5/117) do tecido ovariano e em 2,8% (3/108) do sangue. Em nenhuma das amostras de líquido folicular foi detectado o DNA viral. Nas amostras sorológicas observou-se 83,6% (117/140) de positividade para o BoHV-1. Dentre os animais positivos na PCR, independente das amostras, todos apresentavam positividade no teste de soroneutralização para detecção de anticorpos para BoHV-1. Conclui-se que em animais de diferentes rebanhos analisados foi detectada alta prevalência de anticorpos contra o BoHV-1 e que nos testes moleculares houve a presença do DNA viral em amostras de tecidos ovarianos de bovinos, evidenciando que estas estruturas poderia...(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.[...](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.[...]

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

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

DOENÇA RESPIRATÓRIA BOVINA POR Mycoplasma bovis EM VACAS LACTANTES DE REBANHOS LEITEIROS DE ALTO RENDIMENTO

A ocorrência de doença respiratória bovina (DRB) ocasionada por Mycoplasma bovis foi avaliada em vacas leiteiras em lactação de três rebanhos (A C) de alta produção localizados na mesorregião central oriental do estado do Paraná, sul do Brasil. Com o objetivo de determinar a etiologia de casos clínicos de doença respiratória em vacas em lactação, foi acompanhado por um período de nove meses o rebanho A que apresentou casos recorrentes de DRB em vacas no pós parto. Neste intervalo de tempo, para investigar a etiologia, foram coletados 12 swabs nasais de vacas com quadro clínico de DRB e 12 fragmentos de pulmão de vacas que morreram por DRB. Nos rebanhos B e C, os problemas respiratórios ocorreram de forma diferente. Vacas em pico de lactação apresentaram sinais clínicos agudos de DRB sob a forma de surto comprometendo vários animais simultaneamente. Para o diagnóstico etiológico da DRB foram coletadas amostras de swab nasal em vacas com sinais clínicos de problemas respiratórios agudos no rebanho B (n=20) e no rebanho C (n=9). Todas as amostras foram avaliadas por PCR / RT-PCR para avaliar a presença dos principais agentes etiológicos envolvidos em DRB incluindo Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, vírus respiratório sincicial bovino (BRSV), coronavírus bovino (BCoV), vírus da diarreia viral bovina (BVDV), alfaherpesvírus bovino 1 (BoHV-1) e vírus parainfluenza bovino 3 (BPIV-3). Secções pulmonares de vacas do rebanho A que morreram com BRD foram submetidas à análise histopatológica. Amplicon com tamanho esperado de 488 pb foi obtido por nested-PCR para M. bovis em 9 (75%) dos 12 fragmentos de tecido pulmonar obtidos no rebanho A. Destas, sete vacas apresentaram infecção singular por M. bovis enquanto duas vacas apresentavam infecção mista com P. multocida (n=1) e BVDV (n=1). Nas amostras de swabs nasais (n=12) coletadas do rebanho A, as reações de PCR / RT28 PCR foram positivas para M. bovis (n=3; 25%), P. multocida (n=4; 33,3%), M. haemolytica (n=3; 25%), BVDV (n=2; 16,6%) e BoHV-1 (n=1; 8,3%). Os tecidos pulmonares apresentaram alterações histopatológicas clássicas descritas nos casos de pneumonia por M. bovis em bovinos. Nas vacas do rebanho B foram identificadas apenas infecções com M. bovis (n = 8; 40%) e H. somni (n = 15; 75%). No rebanho C, M. bovis, H. somni e P. multocida foram identificados em swabs nasais de seis (66,6%), duas (22,2%) e três (33,3%) das vacas, respectivamente. Em ambos os rebanhos, nenhum dos outros micro-organismos envolvidos na etiologia de DRB e que foram incluídos nas análises moleculares foi identificado. M. bovis é considerado um micro-organismo fastidioso, com isso, o diagnóstico laboratorial é frequentemente negligenciado e a maioria dos casos clínicos de micoplasmose em bovinos jovens e adultos não é diagnosticada. Este estudo, que envolveu a análise dos principais micro39 organismos causadores de BRD, demonstrou a importância da infecção por M. bovis em vacas adultas em três rebanhos bovinos leiteiros de alta produção. Ressalta-se que, em um rebanho (A), a infecção apresentou-se de forma recorrente, com o surgimento de vários casos clínicos durante os nove meses de avaliação. Em contraposição, nos outros dois rebanhos (B e C) a BRD ocorreu na forma de surto de sinais clínicos respiratórios agudos. Os resultados apontam para a importância de incluir o diagnóstico etiológico de M. bovis em casos de DRB em animais adultos, especialmente em vacas no pós-parto e em vacas no pico lactacional.

The occurrence of bovine respiratory disease (BRD) caused by Mycoplasma bovis was investigated in lactating dairy cows from three high milk yield dairy cattle herds of the eastern central mesoregion of the Paraná state, southern Brazil. With the objective of determining the etiology of clinical cases of respiratory disease in lactating cows, the A herd that presented recurrent cases of DRB in postpartum cows was followed for a period of nine months. In this time interval were collected for determination of the etiology of BRD 12 nasal swabs and 12 lung tissues fragments of cows that died from BRD. Differently, in the other two farms (B and C) the respiratory symptoms in lactating cows, targeting cows at peak lactation, occurred in the form of an outbreak, so several cows presented acute cases of BRD simultaneously. For BRD diagnostic 29 nasal swabs samples were collected in cows with clinical signs of acute respiratory problem in farm B (n=20) and farm C (n=9). All samples were evaluated by PCR / RT-PCR for nucleic acid detection of the main BRD etiological agents including Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoHV-1), and bovine parainfluenza virus 3 (BPIV-3). Pulmonary sections cows that died with BRD were submitted to histopathological analysis. Amplicon with 488 bp length was obtained in nested-PCR for M. bovis in 9 (75%) of the 12 lung tissue fragments obtained from farm A. Of these, seven cows presented M. bovis single infection while two cows had mixed infection with P. multocida (n=1) and BVDV (n=1). In nasal swabs samples (n=12) collected from farm A the PCR / RT-PCR reactions were positive for M. bovis (n=3; 25%), P. multocida (n=4; 33.3%), M. haemolytica (n=3; 25%), BVDV (n=2; 16.6%), and BoHV-1 (n=1; 8.3%). Pulmonary tissues showed classic histopathological changes described in cases of M. bovis pneumonia in cattle. Differently, in cows from farm B were identified only infections with M. bovis (n=8; 40%) and H. somni (n=15; 75%). In farm C the M. bovis, H. somni, and P. multocida were identified in nasal swabs of 6 (66.6%), 2 (22.2%), and 3 (33.3%) cows, respectively. In both herds, none of the other microorganisms involved in BRD included in the analysis were identified. M. bovis is considered a fastidious microorganism. Thus, the laboratory diagnosis is neglected and most clinical cases of mycoplasmosis in cattle are not diagnosed. This study, which involved the analysis of the main microorganisms causing BRD, demonstrated the importance of M. bovis infection in adult cows in three high milk yield dairy cattle herds. It is highlighted that in a herd (A) the infection occurred recurrently, with several cases during 9 months of evaluation. In contrast, in the two other herds (B and C) the BRD occurred as an outbreak of acute respiratory clinical signs. The results point to the importance of including the M. bovis diagnosis in cases of BRD in adult animals, especially in newly calved cows and cows at peak milk yield.

DERMATÓFITOS EM GATOS SEM DERMATOPATIAS NA REGIÃO METROPOLITANA DE FLORIANÓPOLIS SC

O alfaherpesvírus bubalino 1 (BuHV1) e os alfaherpesvírus bovinos 1 (BoHV1), 5 (BoHV5) e são membros da ordem Herpesvirales, família Herpesviridae, subfamília Alphaherpesvirinae, gênero Varicellovirus. O BoHV1 e o BoHV5 são subdivididos em subtipos (BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c). No Brasil circulam BoHV1 e BoHV5 de todos os subtipos até o presente reconhecidos. Em vista dessa variedade de alfaherpesvírus potencialmente infecciosos para bovinos e bubalinos, o conhecimento sobre estes agentes é essencial para a implementação de medidas de controle ou erradicação. Com o objetivo de permitir algumas comparações entre estes agentes e as respostas por eles induzidas em seus hospedeiros, primeiramente foi realizado o sequenciamento do genoma completo de uma amostra de BuHV1 (b6), isolada a partir de prepúcio de búfalos (Bubalus bubalis) em 1972, na Austrália. O objetivo foi disponibilizar a primeira sequência completa de um alfaherpesvírus bubalino e avaliar o grau de similaridade entre o genoma desse vírus e os alfaherpesvírus de bovinos. O genoma sequenciado compreende 137.452 pares de bases (pb), com um nível de similaridade nucleotídica de 92,2% com BoHV5 (SV507/99) e 76,7% com BoHV1 (NVSL). Estes resultados permitirão estudos futuros buscando reconstruir a história evolutiva destes vírus, a importância de infecções interespécies na geração de variantes destes agentes e possíveis associações entre tais infecções e patogenicidade. Na segunda etapa dos estudos, foram realizados diversos testes sorológicos buscando definir uma metodologia de diagnóstico adequada para a identificação de anticorpos contra BuHV1 e todos os diferentes subtipos de BoHV1 e BoHV5. Inicialmente, 600 amostras de soros de bovinos de campo foram testadas através do teste de soroneutralização (SN), realizada frente aos sete alfaherpesvírus em estudo. Os resultados da SN foram influenciados pela escolha do vírus desafio utilizado no ensaio. Para obter a sensibilidade máxima do teste (259/600), foi necessário combinar resultados positivos frente a pelo menos cinco diferentes amostras virais. Em seguida, foram preparados ensaios do tipo ELISA para detecção de anticorpos utilizando antígenos preparados com cada um dos diferentes tipos/subtipos desses vírus (BuHV1; BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c), individualmente (chamado single ELISA ou sAgELISA). Os resultados obtidos foram comparados com os resultados da soroneutralização (SN). A sensibilidade do sAgELISA variou significativamente conforme a amostra viral utilizada no preparo dos antígenos. Considerando os resultados frente a apenas um antígeno, a maior sensibilidade foi de 96,1% (249/259), obtida com apenas uma amostra de BoHV5c. Foram necessários pelo menos seis antígenos (BuHV1; BoHV1.2a, 1.2b; BoHV5a, b, c) para atingir a sensibilidade máxima do teste (263/259). Assim, foi desenvolvido um ELISA combinando vários antígenos em um mesmo teste (chamado multiple ELISA ou mAgELISA). O mAgELISA detectou 263 amostras positivas, resultando em uma concordância ótima entre sAgELISA e mAgELISA (=0,99). Frente a SN, o mAgELISA apresentou uma sensibilidade de 96,5% e especificidade de 96,1% (=0,93; VPP=95,0%; VPN=97,3%). Essas diferenças não foram estatisticamente significativas. Os resultados obtidos com a SN e mAgELISA foram comparados também a um ELISA comercial para a detecção de anticorpos contra BoHV1. O ELISA comercial utilizado nas comparações não apresentou resultados significativamente diferentes dos demais testes realizados, no entanto, revelou o maior número de resultados discrepantes em relação ao SN, considerado padrão-ouro. Estes resultados revelam que o mAgELISA aqui relatado é adequado para a detecção de anticorpos para BuHV1, BoHV1 e BoHV5, com sensibilidade e especificidade significativamente comparáveis às da SN.

Bubaline alphaherpesvirus 1 (BuHV1), Bovine alphaherpesviruses 1 (BoHV1) and 5 (BoHV5) are members of the order Herpesvirales family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. The BoHV1 and BoHV5 are divided into subtypes (BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c). In Brazil, circulates BoHV 1 and BoHV 5 from all subtypes known to the present. In view of this variety of potentially infectious alphaherpesviruses for bovines and buffaloes, knowledge about these agents is essential for the implementation of control or eradication measures. In order to gain a deeper understanding about these agents and the responses induced by them in their hosts, initially , the complete genome sequence of BuHV1-b6, one of the first BuHV1 viruses isolated in Australia in 1972, is reported. The objective was to provide the first complete sequence of a buffalo alphaherpesvirus and to evaluate the degree of similarity between BuHV1 and bovine alphaherpesviruses genomes. The BuHV1-b6 genome is a linear double-stranded DNA molecule, 137,452 bp long, with overall sequence of the 92.2% similarity at the nucleotide level to the reference BoHV5 (SV507/99) strain and 76.7% with BoHV1 (NVSL) strain. These results are expected to be valuable for further studies involving evolutionary chain of these viruses, the interspecies infections importance in generation of variants and possible associations between such infections and pathogenicity. In the second stage of the study, several serological tests were performed in order to define a appropriate diagnostic methodology for the identification of antibodies against BuHV1 and all different subtypes of BoHV1 and BoHV5. Initially, 600 bovine field serum samples were screened in serum neutralization tests (SN) performed against all seven virus types/subtypes. The SN results were influenced by the choice of the challenge virus. The maximum number of positive sera (259/600) was detected by adding the positive results obtained with at least five different viruses. Seven enzyme linked immunoassays were prepared with each of the seven antigens (BuHV1; BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c), individually (single antigen ELISAs; sAgELISAs). The results obtained were compared to the SN. The sensitivity of the sAgELISAs was also influenced by the choice of antigen. Considering the results against only one of them, the best sensitivity was 96.1% (249/259), obtained with BoHV5c. Maximum sAgELISA sensitivity (263/259) was achieved when positive results of at least six viruses (BuHV1; BoHV1.2a, 1.2b; BoHV5a, b, c) were combined. A multiple antigen ELISA (mAgELISA) was then prepared by combining of different viral antigens in the same test. The mAgELISA detected 263 positive samples, resulting an optimum concordance between sAgELISA and mAgELISA (=0.99). When compared to SN, the mAgELISA revealed 96.5% sensitivity and 96.1% specificity (=0.93; PPV=95.0%; NPV=97.3%). These differences were not statistically significant. The results of both SN and mAgELISA were compared to a commercially available (IBRgB) ELISA. The results of the commercial ELISA did not vary significantly in relation to others tests performed, however, revealed the highest number of discrepant results in relation to SN, taken as the gold standard. These results reveal that the mAgELISA reported here is suitable for the detection of antibodies to BuHV1, BoHV1 and BoHV5 with sensitivity and specificity significantly comparable to those of SN.

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

Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006) / Identification and differentiation of herpesvirus types 1 and 5 isolated from clinical samples in central-southern Brazil, Argentina and Uruguay (1987-2006)

Os herpesvírus bovino tipos 1 e 5 (BoHV-1; BoHV-5) são genética e antigenicamente muito semelhantes e por isso são indistinguíveis pela maioria dos testes diagnósticos. Como o BoHV-1 tem sido classicamente associado com doença respiratória e genital, os herpesvírus isolados dessas enfermidades têm sido provisoriamente - e às vezes definitivamente - identificados como BoHV-1. Da mesma forma, os casos de infecção neurológica por herpesvírus em bovinos têm sido atribuídos em sua totalidade ao BoHV-5. Este trabalho relata a identificação de 40 amostras de herpesvírus isoladas de diferentes casos clínicos na região Centro-Sul do Brasil, Argentina e Uruguai entre 1987 e 2006, pelo uso de um PCR capaz de diferenciar esses vírus. As amostras identificadas como BoHV-1 (n=16) foram isoladas de doença respiratória (n=3), balanopostite e/ou vulvovaginite (n=3), do sêmen de touros saudáveis (n=5) e de casos doença neurológica (n=5). As amostras virais identificadas como BoHV-5 (n=24) foram em sua maioria isoladas de doença neurológica (n=21), mas também do sêmen de touros saudáveis (n=2) e do baço de um bezerro com doença sistêmica (n=1). Esses resultados demonstram que tanto o BoHV-1 como o BoHV-5 não estão estritamente associados às suas respectivas síndromes clínicas e que podem estar freqüentemente envolvidos em casos clínicos classicamente atribuídos ao outro vírus. Esses achados também reforçam a necessidade de se identificar corretamente os isolados de herpesvírus para um melhor conhecimento da sua patogenia e epidemiologia.(AU)

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.(AU)

Efeito do dimetildioctadecilamônio na resposta imune humoral e celular de cobaios inoculados com o herpesvírus bovino-1 inativados / Effect of dimethyl dioctadecyl ammonium chloride on the humoral and cellular response to inactivated bovine herpesvirus-1 in guinea pigs

Avaliou-se o potencial adjuvante do cloreto de dimetildioctadecilamônio (DDA cloreto) em estimular as respostas imune humoral e celular, do tipo hipersensibilidade cutânea tardia (DTH), em cobaios que receberam preparações de antígeno contendo o herpesvírus bovino tipo 1 (BHV-1) inativado. Os animais foram vacinados com o BHV-1 em cinco diferentes formulações, representadas pelos grupos: A- adjuvante completo / incompleto de Freund, B- hidróxido de alumínio (Al(OH)3), C- DDA cloreto, D- associação Al(OH)3 / DDA cloreto e E- BHV-1 em solução aquosa sem adjuvante. Os animais do grupo F, grupo-controle negativo, foram inoculados apenas com meio de cultivo celular. Os maiores títulos de anticorpos neutralizantes do BHV-1, expressos em log2, foram obtidos nos grupos D, A e C, com títulos de 6,25, 6,0 e 5,25, respectivamente. Na avaliação da DTH, os maiores aumentos na espessura da dobra da pele foram observados nos grupos A (2,4mm), C (1,8mm) e D (1,1mm). O DDA cloreto, utilizado tanto de forma isolada quanto em associação, determinou a potencialização das respostas imunológicas humoral e celular de cobaios imunizados com o BHV-1 inativado.(AU)

The adjuvanticity of dimethyl dioctadecyl ammonium chloride (DDA chloride) to induce humoral and cell mediated (delayed type hypersensitivity DTH –) immune responses was assessed in guinea pigs that received antigen preparations containing inactivated bovine herpesvirus type 1 (BHV-1). The animals were vaccinated with five different formulations, containing BHV-1 represented by groups: A- complete/incomplete Freund adjuvant, B- aluminum hydroxide (Al(OH)3), C- DDA chloride, D- association Al(OH)3/DDA and E- BHV-1 in aqueous solution without adjuvant. Group F was the negative control group and the animals received only cell culture medium. The higher titers of BHV-1 neutralizing antibodies, expressed in log2, were obtained in groups D, A and C with values of 6.25, 6.0 and 5.25, respectively. In the DTH assessment, the higher increase in skin thickness was observed in group A (2.4mm), C (1.8mm) and D (1.1mm). These results showed that DDA chloride used alone or in association to other substance boosted the humoral and cell mediated immune responses in guinea pigs immunized with inactivated BHV-1.(AU)