Photodisinfection of Alphaherpesvirus 1 in bovine semen.

Reproductive biotechnologies are widely consolidated as a methodology in cattle breeding and have an important impact on the genetic improvement of cattle herds. Semen is an important source of dissemination of pathogenic microorganisms during reproductive procedures. To ensure the sanitary quality of the semen, it is essential to consider the presence of various microorganisms including viruses. One of the main viral agents of reproductive interest is Bovine Alphaherpesvirus 1 (BoHV-1), the etiological agent responsible for bovine rhinotracheitis and vulvovaginitis and frequently associated with reproductive efficiency of matrices and bulls. In artificial insemination centers, semen treatment is generally based only on the use of antibiotics, ignoring the possibility of inactivating other non-bacterial infectious agents. In this context, photodisinfection emerges as a promising alternative to inactivate a wide range of microorganisms, offering a complementary or substitution approach to those conventional semen treatment methods. In this work, we evaluated the use of four halogenated sulfonated porphyrins as potential photosensitizers (PSs) for photodynamic inactivation of Bovine Alphaherpesvirus I (BoHV-1) for bovine semen disinfection. The PSs were synthesized and photophysical parameters, such as UV-Vis absorption spectra and singlet oxygen quantum yield (ΦΔ) were presented. Photoinactivation of BoHV-1 was first shown in cell culture and then confirmed in artificially infected bovine semen and then the phototoxicity of PSs against spermatozoa was evaluated. All PSs were effective in BoHV-1 inactivation; however, the photosensitizer containing two chlorine atoms, showed to be more efficient due to the shorter time required for complete viral inactivation. The slight alterations in sperm kinetics were observed, but remained within those acceptable by regulatory agencies for animal reproduction. Although the methodology used in this work only included bovine semen, we emphasize that the proposed photodisinfection methodology can be adapted and applied to a wide range of biological materials and microorganisms of animal or human interest.

Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Field Evaluation of Commercial Vaccines against Infectious Bovine Rhinotracheitis (Ibr) Virus Using Different Immunization Protocols.

Bovine alphaherpesvirus 1 is ubiquitous in cattle populations and is associated with several clinical syndromes, including respiratory disease, genital disease, infertility and abortions. Control of the virus in many parts of the world is achieved primarily through vaccination with either inactivated or live modified viral vaccines. The objective of this study was to evaluate the performance of four commercially available BoHV-1 vaccines commonly used in Central and South America. Animals were divided into eight groups and vaccinated on days 0 and 30. Groups 1 to 4 received two doses of four different BoHV-1 commercial vaccines (named A to D). Groups 5 and 6 received vaccine D plus a vaccine for either Clostridial or Food-and-Mouth-Disease (FMD), respectively. Group 7 received one dose of two different brands of reproductive vaccines. Serum samples were collected from all animals on days 0, 30 and 60 to evaluate neutralizing and isotype-specific (IgG1 and IgG2) antibodies. Of the four commercial vaccines evaluated, only vaccine A induced neutralizing antibodies to titers ≥ 1:8 in 13/15 (86%) of the animals 60 days post-vaccination. Levels of IgG2 antibody increased in all groups, except for group 2 after the first dose of vaccine B. These results show that only vaccine A induced significant and detectable levels of BoHV-1-neutralizing antibodies. The combination of vaccine D with Clostridial or FMD vaccines did not affect neutralizing antibody responses to BoHV-1. The antibody responses of three of the four commercial vaccines analyzed here were lower than admissible by vaccine A. These results may be from vaccination failure, but means to identify the immune signatures predictive of clinical protection against BoHV-1 in cattle should also be considered.

MANα1-2MAN decorated liposomes enhance the immunogenicity induced by a DNA vaccine against BoHV-1.

New technologies in the field of vaccinology arise as a necessity for the treatment and control of many diseases. Whole virus inactivated vaccines and modified live virus ones used against Bovine Herpesvirus-1 (BoHV-1) infection have several disadvantages. Previous works on DNA vaccines against BoHV-1 have demonstrated the capability to induce humoral and cellular immune responses. Nevertheless, 'naked' DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen-presenting cells (APC). In this work, a DNA-based vaccine encoding the truncated version of BoHV-1 glycoprotein D (pCIgD) was evaluated alone and encapsulated in a liposomal formulation containing LPS and decorated with MANα1-2MAN-PEG-DOPE (pCIgD-Man-L). The vaccinations were performed in mice and bovines. The results showed that the use of pCIgD-Man-L enhanced the immune response in both animal models. For humoral immunity, significant differences were achieved when total antibody titres and isotypes were assayed in sera. Regarding cellular immunity, a significant increase in the proliferative response against BoHV-1 was detected in animals vaccinated with pCIgD-Man-L when compared to the response induced in animals vaccinated with pCIgD. In addition, upregulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with the vaccine formulations. When viral challenge was performed, bovines vaccinated with MANα1-2MAN-PEG-DOPE elicited better protection which was evidenced by a lower viral excretion. These results demonstrate that the dendritic cell targeting using MANα1-2MAN decorated liposomes can boost the immunogenicity resulting in a long-lasting immunity. Liposomes decorated with MANα1-2MAN-PEG-DOPE were tested for the first time as a DNA vaccine nanovehicle in cattle as a preventive treatment against BoHV-1. These results open new perspectives for the design of vaccines for the control of bovine rhinotracheitis.

An Improved DNA Vaccine Against Bovine Herpesvirus-1 Using CD40L and a Chemical Adjuvant Induces Specific Cytotoxicity in Mice.

Bovine herpesvirus-1 (BoHV-1) uses many mechanisms to elude the immune system; one of them is spreading intracellularly, even in the presence of specific antiviral antibodies. Cytotoxic T lymphocytes (CTLs) are necessary to eliminate the virus. The main preventive strategy is vaccination based on inactivated virus. These vaccines are poor inducers of cellular immune responses, and complicate serological diagnosis and determination of the real prevalence of infection. DNA vaccines are a good option because of the capacity of Differentiating Infected from Vaccinated Animals-(DIVA vaccine)-and may be the best way to induce cytotoxic responses. Although this type of vaccines leads to only weak "in vivo" expression and poor immune responses, incorporation of molecular and/or chemical adjuvants can improve the latter, both in magnitude and in direction. In this study, we have investigated the specific immune responses elicited in mice by DNA vaccines based on the BoHV-1 glycoprotein D (pCIgD) with and without two different adjuvants: a plasmid encoding for murine CD40L (pCD40L) or Montanide™ 1113101PR (101). Mice vaccinated with pCIgD+CD40L, pCIgD+101, and pCIgD+CD40L+101 developed significantly higher specific antibody titers against BoHV-1 than the pCIgD group (p < 0.01). The animals vaccinated with pCgD+pCD40L+101 raised significantly higher levels of IgG2a and IgG2b (p < 0.01 and p < 0.001, respectively) than mice vaccinated with pCIgD alone. On the contrary, when the activity of CTL against cells infected with BoHV-1 was measured, the vaccine pCgD+pCD40L+101 induced significantly higher levels of cytotoxicity activity (p < 0.001) than pCIgD alone. A significant increase in the CD4+ populations in the group receiving pCIgD+CD40L+101 in comparison with the pCIgD group was observed and, also, interferon gamma, interleukin (IL)-6, and IL-17A levels were higher. Considering the results obtained from this study for humoral and cellular responses in mice, the inclusion of pCD40L and 101 as adjuvants in a BoHV-1 DNA vaccine for cattle is highly recommendable.

Immunoconjugates to increase photoinactivation of bovine alphaherpesvirus 1 in semen.

Artificial insemination and in vitro embryo production are increasingly used to improve the reproductive efficiency of herds, however success of these techniques depends on the sanitary quality of the semen. Insemination centers commonly use antibiotics in their routine procedure, but they are not able against viruses. In this paper, we demonstrate a new approach for disinfecting virus in bovine semen using photoimmunoinactivation, an adaptation of the photodynamic inactivation (PDI) methodology. The photosensitizers (PSs), hematoporphyrin (HP) and zinc tetracarboxy-phthalocyanine (ZnPc) were conjugated to Immunoglobulin Y (IgY) anti-bovine alphaherpesvirus 1 (BoHV-1) and used for PDI against the BoHV-1 viruses in cell culture and compared to the unconjugated PSs. Both treatments proved to be efficient, but a significant decrease in the irradiation time required to completely eliminate the virus was observed in the samples treated with the immunoconjugates. Photophysical measurements help us to understand the coupling between PSs and IgY and the evaluated production of singlet oxygen. Following the cell culture test, the same approach was applied in semen artificially infected with BoHV-1. The immunoconjugates were also efficient for complete virus inactivation up to 5 min of irradiation and proved to be safe using several parameters of sperm viability, demonstrating the feasibility of our strategy for disinfection viruses in semen.

Mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows.

The etiology and pathologic findings of bovine respiratory disease (BRD) in adult dairy cows (n = 35) from a commercial dairy herd in Southern Brazil were investigated. Pulmonary samples were examined for histopathologic patterns and specific features within these patterns, while immunohistochemical (IHC) assays were designed to detect the intralesional antigens of viral infectious disease agents and Mycoplasma bovis. Pneumonia was diagnosed in 91.4% (32/35) of these cases; neither pneumonia nor any of the infectious disease pathogens evaluated occurred in three cows. The presence of multiple respiratory pathogens in 75% (24/32) of these cases indicated the complex origin of pneumonia in cattle. Interstitial pneumonia, necrosuppurative bronchopneumonia and suppurative bronchopneumonia were the principal patterns of pulmonary disease identified by histopathology. The most frequent pathogens identified by IHC were bovine viral diarrhea virus (BVDV; n = 18), M. bovis (n = 16) and bovine alphaherpesvirus type 1 (BoHV-1; n = 14), followed by bovine respiratory syncytial virus (BRSV; n = 11) and bovine parainfluenza virus type 3 (BPIV-3; n = 5). Obliterative bronchiolitis and peribronchial lymphocytic cuffings were the characteristic histopathologic features associated with M. bovis. Necrohemorrhagic bronchitis with bronchial angiogenesis was associated with BoHV-1. Necrotizing bronchitis and bronchiolitis were associated with BVDV, BoHV-1 and BRSV. Ballooning degeneration of the bronchial and bronchiolar epithelia was associated with BRSV and BoHV-1. This is the first report from Brazil that correlated the histopathologic findings of BRD with the associated infectious disease agents by immunohistochemistry. M. bovis was frequently detected in the tissues of cows with fatal pulmonary disease during this study and may be a possible primary disease pathogen associated with the development of BRD in dairy cows. Additionally, the histopathologic features identified within patterns of pulmonary disease during this investigation may be an efficient diagnostic tool to associate histopathologic findings with specific agents of BRD in dairy cows.

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 / 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)

Infecção latente pelo herpesvírus bovino tipo 1 em búfalos (Bubalus bubalis) no Rio Grande do Sul / Bovine herpesvirus type 1 latent infection in buffalo (Bubalus bubalis) in Rio Grande do Sul

Apesar dos bovinos serem considerados os hospedeiros naturais do BoHV-1, estudos sorológicos têm sugerido que búfalos podem ser suscetíveis ao BoHV-1 e a outros alfa-herpesvírus geneticamente relacionados. O objetivo deste estudo foi detectar a presença de DNA viral de BoHV-1 em 202 amostras de gânglios trigêmeos de búfalos, pela técnica de semi-nested PCR, para detecção de um segmento do gene codificante da glicoproteína D (gD) do BoHV-1. Além disso, 242 amostras de soro foram analisadas pela técnica de soroneutralização (SN) para a detecção de anticorpos neutralizantes contra BoHV-1, BoHV-5 e BuHV. Todas as amostras clínicas foram coletadas em um matadouro na cidade de Pelotas, RS, Brasil. O DNA de BoHV-1 foi detectado em 61 (30,1%) gânglios, e os resultados da SN demonstraram que 27,6% dos animais apresentaram anticorpos contra, pelo menos, um dos vírus testados. O sequenciamento genômico e a análise de 14 amplicons confirmaram a presença do DNA do BoHV-1 nos tecidos analisados. Em resumo, os resultados indicam que o BoHV-1 está distribuído em rebanhos bubalinos provenientes da região Sul do Brasil. Entretanto, são necessárias investigações adicionais, no sentido de elucidar o papel exato dos búfalos na epidemiologia das infecções pelo BoHV-1.(AU)

Although bovines are natural hosts for BoHV-1, serologic studies in several countries have suggested that buffaloes (Bubalus bubalis) may be susceptible to BoHV-1 and other genetically related alphaherpesvirus. This study aimed to investigate the presence of BoHV-1 DNA in trigeminal ganglia from 202 buffaloes by a semi-nested PCR to amplify partially the glycoprotein D (gD) gene of BoHV-1. Additionally, 242 serum samples were tested by serum neutralization (SN) for the detection of antibodies against BoHV-1, BoHV-5 and BuHV. All clinical samples were collected in a slaughterhouse located in Pelotas, RS, Brazil. BoHV-1 DNA was detected in 61 (30.1%) of the samples and SN revealed 27.6% of the animals with neutralizing antibodies against at least one of the tested viruses. Nucleotide sequencing of 15 amplicons followed by BLAST analysis confirmed the presence of BoHV-1 DNA in the analyzed tissues. Taken together, these data indicate that BoHV-1 infection is distributed in buffaloes in southern Brazil. However, the role of buffaloes in the BoHV-1 epidemiology needs further investigation.(AU)

Infecção latente pelo herpesvírus bovino tipo 1 em búfalos (Bubalus bubalis) no Rio Grande do Sul / Bovine herpesvirus type 1 latent infection in buffalo (Bubalus bubalis) in Rio Grande do Sul

Apesar dos bovinos serem considerados os hospedeiros naturais do BoHV-1, estudos sorológicos têm sugerido que búfalos podem ser suscetíveis ao BoHV-1 e a outros alfa-herpesvírus geneticamente relacionados. O objetivo deste estudo foi detectar a presença de DNA viral de BoHV-1 em 202 amostras de gânglios trigêmeos de búfalos, pela técnica de semi-nested PCR, para detecção de um segmento do gene codificante da glicoproteína D (gD) do BoHV-1. Além disso, 242 amostras de soro foram analisadas pela técnica de soroneutralização (SN) para a detecção de anticorpos neutralizantes contra BoHV-1, BoHV-5 e BuHV. Todas as amostras clínicas foram coletadas em um matadouro na cidade de Pelotas, RS, Brasil. O DNA de BoHV-1 foi detectado em 61 (30,1%) gânglios, e os resultados da SN demonstraram que 27,6% dos animais apresentaram anticorpos contra, pelo menos, um dos vírus testados. O sequenciamento genômico e a análise de 14 amplicons confirmaram a presença do DNA do BoHV-1 nos tecidos analisados. Em resumo, os resultados indicam que o BoHV-1 está distribuído em rebanhos bubalinos provenientes da região Sul do Brasil. Entretanto, são necessárias investigações adicionais, no sentido de elucidar o papel exato dos búfalos na epidemiologia das infecções pelo BoHV-1.(AU)

Although bovines are natural hosts for BoHV-1, serologic studies in several countries have suggested that buffaloes (Bubalus bubalis) may be susceptible to BoHV-1 and other genetically related alphaherpesvirus. This study aimed to investigate the presence of BoHV-1 DNA in trigeminal ganglia from 202 buffaloes by a semi-nested PCR to amplify partially the glycoprotein D (gD) gene of BoHV-1. Additionally, 242 serum samples were tested by serum neutralization (SN) for the detection of antibodies against BoHV-1, BoHV-5 and BuHV. All clinical samples were collected in a slaughterhouse located in Pelotas, RS, Brazil. BoHV-1 DNA was detected in 61 (30.1%) of the samples and SN revealed 27.6% of the animals with neutralizing antibodies against at least one of the tested viruses. Nucleotide sequencing of 15 amplicons followed by BLAST analysis confirmed the presence of BoHV-1 DNA in the analyzed tissues. Taken together, these data indicate that BoHV-1 infection is distributed in buffaloes in southern Brazil. However, the role of buffaloes in the BoHV-1 epidemiology needs further investigation.(AU)

Bovine alphaherpesvirus 1 and 5 in semen from bulls presenting genital lesions under field conditions in Brazil / Alfaherpesvírus bovino 1 e 5 em sêmen de touros com lesões genitais, sob condições a campo no Brasil

Bovine alphaherpesviruses 1 and 5 (BoHV-1/5) are main pathogens of respiratory, reproductive and neurological diseases in cattle. The aim of this study was to investigate the frequency of neutralizing antibodies against BoHV-1/5 in serum samples and to detect viral DNA in semen of bulls from beef cattle farms located in RS. A total of 372 serum and semen sample from bulls were collected in eighteen farms. Serum samples were submitted to virus neutralization (VN) assay, while semen samples were used to detect BoHV-1 and BoHV-5 DNA by PCR. VN results showed that BoHV-1/5 antibodies were detected in bulls of 66.7% (12/18) of the farms, 295 (79.5%) BoHV positive bulls, 287 for BoHV-1 and 234 for BoHV-; at 43 vaccinated bulls 72.1% (31/43) showing serology negative. BoHV-1/5 DNA was detected in the semen of three bulls; one of the them presenting BoHV-1, one out three presenting BoHV-5 and one BoHV-1/5.co-infection All BoHV DNA positive samples came from animals presenting posthitis and other genital lesions at sampling. Results showed a high seroprevalence of BoHV-1/5 antibodies in bulls as well as strong evidence that these viruses are actively circulating in the cattle farms. A remarkable finding is that in the presence of clinically evident lesions in the genital tract, both BoHV-1 and 5 may found in semen.(AU)

Os alfa-herpesvírus bovinos 1 e 5 (BoHV-1/5) são importantes patógenos de doença respiratória, reprodutiva e neurológica em bovinos. O objetivo deste estudo foi investigar a frequência de detecção de anticorpos neutralizantes contra BoHV-1/5 em amostra de soro e detectar DNA viral em sêmen de touros do rebanho bovino localizado nas fazendas de gado de corte do RS. Um total de 371 amostras de soro e sêmen foi coletado de touros em 18 fazendas, 325 das quais são provenientes de touros não vacinados e 43 de vacinados. Amostras de soro foram submetidas à técnica de vírus-neutralização (VN), enquanto as amostras de sêmen foram submetidas à extração de DNA e posterior PCR (polymerase chain reaction) para detecção de BoHV-1 e 5. Os resultados da VN demostraram que anticorpos contra BoHV-1/5 foram detectados nos touros não vacinados em 66,7% (12/18) das fazendas, 295 (79,5%) touros mostraram-se positivos para BoHV, 287 para BoHV-1 e 234 para BoHV-5; e para 43 touros vacinados, observou-se que 72,1% (31/43) foram negativos na sorologia DNA de BoHV-1/5, detectado no sêmen de três touros: um deles apresentava BoHV-1, outro BoHV-5 e em um foi detectada coinfecção por BoHV-1/5. Todas as amostras positivas para o DNA viral eram provenientes de animais que apresentavam lesões de postite e outras lesões genitais. Esses resultados demonstram que há uma alta soroprevalência de BoHV-1/5 em touros, bem como uma forte evidência de que esses vírus estão circulando ativamente no rebanho bovino dessas fazendas. Um achado interessante foi a detecção de BoHV-1 e 5 em touros com lesões na região do trato genital.(AU)

Bovine alphaherpesvirus 1 and 5 in semen from bulls presenting genital lesions under field conditions in Brazil / Alfaherpesvírus bovino 1 e 5 em sêmen de touros com lesões genitais, sob condições a campo no Brasil

Bovine alphaherpesviruses 1 and 5 (BoHV-1/5) are main pathogens of respiratory, reproductive and neurological diseases in cattle. The aim of this study was to investigate the frequency of neutralizing antibodies against BoHV-1/5 in serum samples and to detect viral DNA in semen of bulls from beef cattle farms located in RS. A total of 372 serum and semen sample from bulls were collected in eighteen farms. Serum samples were submitted to virus neutralization (VN) assay, while semen samples were used to detect BoHV-1 and BoHV-5 DNA by PCR. VN results showed that BoHV-1/5 antibodies were detected in bulls of 66.7% (12/18) of the farms, 295 (79.5%) BoHV positive bulls, 287 for BoHV-1 and 234 for BoHV-; at 43 vaccinated bulls 72.1% (31/43) showing serology negative. BoHV-1/5 DNA was detected in the semen of three bulls; one of the them presenting BoHV-1, one out three presenting BoHV-5 and one BoHV-1/5.co-infection All BoHV DNA positive samples came from animals presenting posthitis and other genital lesions at sampling. Results showed a high seroprevalence of BoHV-1/5 antibodies in bulls as well as strong evidence that these viruses are actively circulating in the cattle farms. A remarkable finding is that in the presence of clinically evident lesions in the genital tract, both BoHV-1 and 5 may found in semen.(AU)

Os alfa-herpesvírus bovinos 1 e 5 (BoHV-1/5) são importantes patógenos de doença respiratória, reprodutiva e neurológica em bovinos. O objetivo deste estudo foi investigar a frequência de detecção de anticorpos neutralizantes contra BoHV-1/5 em amostra de soro e detectar DNA viral em sêmen de touros do rebanho bovino localizado nas fazendas de gado de corte do RS. Um total de 371 amostras de soro e sêmen foi coletado de touros em 18 fazendas, 325 das quais são provenientes de touros não vacinados e 43 de vacinados. Amostras de soro foram submetidas à técnica de vírus-neutralização (VN), enquanto as amostras de sêmen foram submetidas à extração de DNA e posterior PCR (polymerase chain reaction) para detecção de BoHV-1 e 5. Os resultados da VN demostraram que anticorpos contra BoHV-1/5 foram detectados nos touros não vacinados em 66,7% (12/18) das fazendas, 295 (79,5%) touros mostraram-se positivos para BoHV, 287 para BoHV-1 e 234 para BoHV-5; e para 43 touros vacinados, observou-se que 72,1% (31/43) foram negativos na sorologia DNA de BoHV-1/5, detectado no sêmen de três touros: um deles apresentava BoHV-1, outro BoHV-5 e em um foi detectada coinfecção por BoHV-1/5. Todas as amostras positivas para o DNA viral eram provenientes de animais que apresentavam lesões de postite e outras lesões genitais. Esses resultados demonstram que há uma alta soroprevalência de BoHV-1/5 em touros, bem como uma forte evidência de que esses vírus estão circulando ativamente no rebanho bovino dessas fazendas. Um achado interessante foi a detecção de BoHV-1 e 5 em touros com lesões na região do trato genital.(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.

Soroprevalência de herpesvírus bovino tipo 1 em fêmeas bovinas na microrregião do Vale do Rio dos Bois, Goiás, Brasil / Seroprevalence of bovine herpesvirus type 1 in bovine females in the microregion of the Vale do Rio dos Bois, Goias, Brazil

Livestock is one of the main activities of the gross domestic Product (GDP), exercising a fundamental role in the land use and occupation process in Brazil. Infectious diseases, bacterial, viral or parasitic origin, may affect the reproductive system of both males and females, causing a series of problems of infertility with different levels of occurrence. One of the most important diseases that affect the reproductive sphere of viral origin and that have already been identified in cattle in Brazil the infectious bovine rhinotracheitis (IBR). The aim of this work was to carry out an epidemiological study in the State of Goiás, specifically in the Vale do Rio dos Bois microregion (municipalities of Cezarina, Mairipotaba and Pontalina), detecting the presence of antibodies to the IBR, by vírusneutralização (VN). The properties studied were found a seroprevalence of 96.4% for neutralizing antibodies against IBR in bovine females over 24 months. The results found in this study showed that there is a high prevalence of infection for BoHV-1 at Vale do Rio dos Bois microregion. The data for these studies can serve as a base to plot strategies for the control of the disease, with the guidance of producers.

A pecuária constitui uma das principais atividades econômicas contribuintes para o Produto Interno Bruto (PIB) brasileiro, exercendo papel fundamental no processo de uso e ocupação de terras no Brasil. Doenças infecciosas de origem bacteriana, viral ou parasitária podem afetar o sistema reprodutivo, tanto dos machos como das fêmeas, causando uma série de problemas de infertilidade nos rebanhos, com diferentes níveis de gravidade. Dentre as doenças mais importantes de origem viral que afetam a natureza reprodutiva e que já foram identificadas em bovinos no Brasil, destaca-se a Rinotraqueíte Infecciosa Bovina (IBR). O objetivo deste trabalho foi realizar um estudo epidemiológico no estado de Goiás, especificamente na Microrregião do Vale do Rio dos Bois (municípios de Cezarina, Mairipotaba e Pontalina), detectando a presença de anticorpos contra a IBR pelo teste de vírus-neutralização (VN). Das propriedades estudadas, foi encontrada uma soroprevalência de 96,4% para anticorpos neutralizantes contra a IBR em fêmeas bovinas acima de 24 meses. Os resultados encontrados no presente estudo demonstraram que há uma elevada taxa de prevalência da infecção por BoHV-1 na Microrregião do Vale do Rio dos Bois. Os dados deste estudo poderão servir de base para se traçarem estratégias para o controle da doença, com orientação aos produtores.

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)

Soroprevalência de herpesvírus bovino tipo 1 em fêmeas bovinas na microrregião do Vale do Rio dos Bois, Goiás, Brasil / Seroprevalence of bovine herpesvirus type 1 in bovine females in the microregion of the Vale do Rio dos Bois, Goias, Brazil

Livestock is one of the main activities of the gross domestic Product (GDP), exercising a fundamental role in the land use and occupation process in Brazil. Infectious diseases, bacterial, viral or parasitic origin, may affect the reproductive system of both males and females, causing a series of problems of infertility with different levels of occurrence. One of the most important diseases that affect the reproductive sphere of viral origin and that have already been identified in cattle in Brazil the infectious bovine rhinotracheitis (IBR). The aim of this work was to carry out an epidemiological study in the State of Goiás, specifically in the Vale do Rio dos Bois microregion (municipalities of Cezarina, Mairipotaba and Pontalina), detecting the presence of antibodies to the IBR, by vírusneutralização (VN). The properties studied were found a seroprevalence of 96.4% for neutralizing antibodies against IBR in bovine females over 24 months. The results found in this study showed that there is a high prevalence of infection for BoHV-1 at Vale do Rio dos Bois microregion. The data for these studies can serve as a base to plot strategies for the control of the disease, with the guidance of producers.(AU)

A pecuária constitui uma das principais atividades econômicas contribuintes para o Produto Interno Bruto (PIB) brasileiro, exercendo papel fundamental no processo de uso e ocupação de terras no Brasil. Doenças infecciosas de origem bacteriana, viral ou parasitária podem afetar o sistema reprodutivo, tanto dos machos como das fêmeas, causando uma série de problemas de infertilidade nos rebanhos, com diferentes níveis de gravidade. Dentre as doenças mais importantes de origem viral que afetam a natureza reprodutiva e que já foram identificadas em bovinos no Brasil, destaca-se a Rinotraqueíte Infecciosa Bovina (IBR). O objetivo deste trabalho foi realizar um estudo epidemiológico no estado de Goiás, especificamente na Microrregião do Vale do Rio dos Bois (municípios de Cezarina, Mairipotaba e Pontalina), detectando a presença de anticorpos contra a IBR pelo teste de vírus-neutralização (VN). Das propriedades estudadas, foi encontrada uma soroprevalência de 96,4% para anticorpos neutralizantes contra a IBR em fêmeas bovinas acima de 24 meses. Os resultados encontrados no presente estudo demonstraram que há uma elevada taxa de prevalência da infecção por BoHV-1 na Microrregião do Vale do Rio dos Bois. Os dados deste estudo poderão servir de base para se traçarem estratégias para o controle da doença, com orientação aos produtores.(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)

Photodynamic inactivation of Bovine herpesvirus type 1 (BoHV-1) by porphyrins.

In this work, the photodynamic efficiency of anionic meso-tetrakis sulfonophenyl (TPPS4), cationic meso-tetrakis methylpyridiniumyl (TMPyP) and their zinc complexes (ZnTPPS4 and ZnTMPyP) in the inactivation of Bovine herpesvirus type 1 (BoHV-1) was evaluated. At a non-cytotoxic concentration, all porphyrins showed significant antiviral activity after irradiation using a halogen lamp. The efficiency of the cationic porphyrins was higher than that of the anionic ones. Porphyrin complexation with zinc increases its lipophilicity and the number of absorbed photons, dramatically reducing the time for complete virus inactivation. The high superposition of the compound optical absorption and light source emission spectra played a key role in the virus inactivation efficiency. The results demonstrated the high effectivity of the photodynamic inactivation of BoHV-1. This method can be used as an auxiliary in the treatment of disorders attributed to BoHV-1 infection, and the porphyrins are promising photosensitizers for this application.

Serological investigation of antibodies against respiratory viruses in calves from Brazilian family farming and their relation to clinical signs of bovine respiratory disease / Investigação sorológica de anticorpos contra vírus respiratórios em bezerros de propriedades familiares brasileiras e sua relação com os sinais clínicos da doença respiratória dos bovinos

Bovine respiratory disease (BRD) is responsible for economic losses in cattle production. Viruses are categorized as primary etiological agents. The aims of this study were to evaluate the presence of antibodies against bovine viral diarrhea virus (BVDV), bovine herpes virus type 1 (BoHV-1), and bovine respiratory syncytial virus (BRSV) in healthy and BRD calves from family farming in relation to clinical signs of BRD. Hundred and forty-five calves were randomly selected and physical examination was performed. Only 123 animals were classified as healthy and BRD calves. Antibodies were evaluated by virus neutralization test. Person's Chi-square test and Fisher's exact test were performed as univariate analysis. Binary Logistic Regression was applied as multivariate analysis. Variables with P<0.10 were considered statistically significant. Variables with 0.15

A doença respiratória dos bovinos (DRB) é responsável por importantes perdas econômicas para a produção bovina, com maior impacto na agricultura familiar. Os vírus são comumente caracterizados como agentes etiológicos primários devido a mudanças na mucosa respiratória, na produção de citocinas e no funcionamento do sistema imune. Os objetivos deste estudo transversal foram avaliar a presença de anticorpos contra o vírus da diarreia viral bovina (VDVB), herpes vírus bovino tipo 1 (HVbo-1) e vírus respiratório sincicial bovino (VRSB) em bezerros sadios e com DRB de assentamentos em associação com a presença sinais clínicos de DRB. Cento e quarenta e cinco animais foram randomicamente selecionados e exame físico foi realizado. Somente 123 animais foram classificados em sadios e com DRB. Anticorpos foram identificados pelo teste de virusneutralização. Teste de qui-quadrado e teste exato de Fisher foram utilizados como análise univariada. A análise multivariada foi realizada pela regressão binária logística com o método Backward conditional. Variáveis com P<0,10 foram considerados significantes. Variáveis com 0,15