Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus / 生物工程学报

Three pairs of primers were designed according to the conserved region of IBRV gB gene published in GenBank(GenBank Accession No. DQ006857.1) using the software Primer Explorer V4. The loop mediated isothermal amplification (LAMP) assay was established by optimization of the reaction system and then evaluated through sensitivity and specificity tests. In total 393 clinical specimens were detected for IBRV using the established LAMP assay performed at 65℃ for 50 min, which produced a ladder-like pattern of amplification bands and the detection result could be judged by color change. The sensitivity of the assay was 10 copies/μL plasmid DNA which was 1000 times higher than that by PCR method and equivalent to nested-PCR. There was no cross-reactivity of the assay with bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and vesicular stomatitis virus (VSV). The positive rate of 301 nasal swabs and 92 serum specimens were 87.6% and 58.8%, respectively, which meant nasal swab specimen was more suitable for clinical IBRV detection by the method. The IBRV LAMP method established in this study has the advantages of visualization, quickness, specificity and sensitivity and be suitable for rapid detection of clinical IBRV detection on the spot.

Presence of IgG antibodies against reproductive infections in breeding bulls of Magdalena Medio, Colombia / Presencia de anticuerpos tipo IgG contra infecciones reproductivas en toros reproductores de la región del Magdalena Medio, Colombia / Presença de anticorpos IgG contra infecções reprodutivas de touros reprodutores do Magdalena Médio, Colômbia

Background: cattle farming is an important economic activity in several regions of Colombia. However, farmers are unaware of their cattle health status, and particularly of bacterial, parasitic, and viral diseases, which affect reproduction and profitability. Objective: to assess the presence of IgG antibodies against infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), bovine leukosis, leptospirosis, brucellosis, and Neospora caninum in breeding bulls in the Magdalena Medio of Colombia, according to age, breed, and origin. Methods: a descriptive, cross-sectional study was conducted with 174 male breeding cattle from several subspecies. Animals were above 18 months of age and belonged to dual-purpose farms in the Magdalena Medio region. Presence of IgG anti-IBR, BVD, brucellosis, neosporosis, and leukosis antibodies were determined with an enzyme-linked immunosorbent assay (ELISA). A sample of 156 males was assessed for leptospirosis using a microscopic agglutination test (MAT) in a dark field. Results: IgG anti-IBR antibodies were found in 92.5% of the animals, anti-BVD in 62.6%, anti-Neospora caninum in 79.3%, anti-Brucella abortus in 4.02%, anti-Leptospira pomona in 8.33%, and anti-L. hardjo in 15.38%, with >1:100 titres. Besides, 64.94% prevalence was observed for bovine leukosis. Conclusions: a high prevalence of IgG anti-IBR, IgG anti-BVD, IgG anti- Neospora caninum, IgG anti-leukosis, and IgG anti-lepstospirosis antibodies were observed in Magdalena Medio bulls. With respect to B. abortus, confirmatory and competitive ELISA test should be performed and sanitary standards need to be established. Detection of IgG antibodies among bulls reveals poor sanitary conditions, absent of protective-vaccination antibodies and an urgent need for health control measurements.

Antecedentes: la ganadería bovina es una importante actividad económica desarrollada en varias regiones del país, sin embargo, los ganaderos desconocen el estado sanitario de los bovinos con respecto a enfermedades bacterianas, parasitarias y virales, lo que afecta la reproducción y la rentabilidad. Objetivo: evidenciar la presencia de anticuerpos tipo IgG anti-rinotraqueitis infecciosa bovina (IBR), diarrea viral bovina (BVD), leucosis bovina, leptospirosis, brucelosis y Neospora caninum en los toros reproductores del Magdalena Medio colombiano, en asociación con variables de edad, raza y procedencia. Métodos: se realizó un estudio descriptivo de corte transversal en un grupo poblacional de 174 machos reproductores bovinos de diferentes subespecies. Los animales eran mayores de 18 meses en fincas destinadas a ganadería doble propósito ubicadas en la región del Magdalena Medio. Mediante un ensayo de inmunoabsorción enzimática (ELISA) se determinó la presencia de anticuerpos tipo IgG anti-IBR, BVD, brucelosis, neosporosis y leucosis. Para el caso de la leptospirosis, a 156 de los machos se les realizó la prueba de microaglutinación en campo oscuro (MAT). Resultados: se hallaron anticuerpos tipo IgG anti-IBR en un 92,5%, anti-BVD en el 62,6%, anti-Neospora caninum 79,3%, anti-Brucella abortus 4,02%, anti-Leptospira pomona 8,33% y anti-L. hardjo 15,38%, con títulos >1:100. Además, para leucosis bovina se obtuvo una prevalencia del 64,94%. Conclusión: se observaron altos porcentajes de casos con presencia de anticuerpos IgG anti-IBR, BVD, N. caninum, leucosis y lepstospirosis en el ganado bovino del Magdalena Medio colombiano. Para B. abortus debe realizarse la prueba confirmativa ELISA competitiva y aplicar la norma sanitaria. La determinación de anticuerpos tipo IgG permitió conocer el delicado estado sanitario de los reproductores bovinos, teniendo en cuenta que no han recibido ningún esquema de vacunación y se hace necesario un adecuado control sanitario.

Antecedentes: a pecuária é uma atividade econômica importante em várias regiões do país, no entanto, os agricultores não têm conhecimento do estado de saúde do gado em relação adoenças bacterianas, parasitárias e virais. É por esta razão que as perdas econômicas aumentam e diminuem a qualidade de reprodução. Objetivo: demonstrar a presença de anticorpos IgG para rinotraqueíte-viral bovina (IBR), diarréia viral bovina (BVD), leucose bovina, leptospirose, brucelose e Neospora caninum. Nos machos reprodutores do Magdalena Medio tendo em conta as variáveis idade, raça e origens. Métodos: i foi realizada estudo descritivo transversal em duas populações de 174 machos reprodutores de diferentes subespécies de gado com mais de 18 meses de edad procedentes de fazendas utilizadas para a pecuária de duplo propósito, localizadas na região do Madalena Médio. Para detectar a presença de anticorpos IgG anti-IBR, BVD, brucelose, e enzoótica, neosporose foi determinada pela técnica de ensaio imunoenzimático (ELISA). Para o caso de leptospirose foi realizado teste de microaglutinação em campo escuro (MAT). Resultados: anticorpos IgG anti-IBR foram encontrados em 92,5%, anti-BVD num 62,6%, anti-Neospora caninum num 79,3%, anti-Brucella abortus 4,02%, anti-Leptospira pomona num 8,33% e anti-L. hardjo 15,38% com títulos > 1:100. Para leucose bovina, uma percentagem de 64,94%, foi obtido. Conclusão: foram observados altos percentagens de casos na presença de anticorpos IgG anti-IBR, BVD, N. caninum, lepstospirosis e leucose nos bovinos do Madalena Médio colombiano, o teste confirmatorio ELISA competitivo. Para a determinação de B. abortus deve ser realizado e dar cumplimento a normatividade sanitaria. A determinação de anticorpos IgG tem permitido conhecer o delicado estado sanitario dos reprodutores bovinos, considerando que eles não receberam qualquer vacinação é necessário um adequado controle de saúde.

Computational prediction of MHC class I epitopes for most common viral diseases in cattle (Bos taurus).

Viral diseases like foot-and-mouth disease (FMD), calf scour (CS), bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR) etc. affect the growth and milk production of cattle (Bos taurus) causing severe economic loss. Epitope-based vaccine designing have been evolved to provide a new strategy for therapeutic application of pathogen-specific immunity in animals. Therefore, identification of major histocompatibility complex (MHC) binding peptides as potential T-cell epitopes is widely applied in peptide vaccine designing and immunotherapy. In this study, MetaMHCI tool was used with seven different algorithms to predict the potential T-cell epitopes for FMD, BVD, IBR and CS in cattle. A total of 54 protein sequences were filtered out from a total set of 6351 sequences of the pathogens causing the said diseases using bioinformatics approaches. These selected protein sequences were used as the key inputs for MetaMHCI tool to predict the epitopes for the BoLA-A11 MHC class I allele of B. taurus. Further, the epitopes were ranked based on a proposed principal component analysis based epitope score (PbES). The best epitope for each disease based on its predictability through maximum number of predictors and low PbES was modeled in PEP-FOLD server and docked with the BoLA-A11 protein for understanding the MHC-epitope interaction. Finally, a total of 78 epitopes were predicted, out of which 27 were for FMD, 25 for BVD, 12 for CS and 14 for IBR. These epitopes could be artificially synthesized and recommended to vaccinate the cattle for the considered diseases. Besides, the methodology adapted here could also be used to predict and analyze the epitopes for other microbial diseases of important animal species.

Surveillance of neutralizing antibodies against Bovine Herpesvirus 1 in cattle herds from different farming property systems / Distribuição de anticorpos neutralizantes contra o Herpesvirus bovino 1 em propriedades bovinas de diferentes sistemas de criação

The aim of this study was to investigate the presence of neutralizing antibodies against bovine herpesvirus 1 (BoHV-1) in 722 non-vaccinated animals from eight properties that use different farming systems (extensive, semi-confinement and confinement). Serum samples were subjected to neutralization tests in order to search for anti-BoHV-1 antibodies. Serological results were categorized as negative, low titer, intermediate titer or high titer. One property showed no positive samples; while other properties presented frequency of positive samples ranging from 17.95% to 86.96%. For animals raised under confinement, the number of positive samples and neutralizing antibody titers were lower compared to others, possibly due to good sanitary practices adopted by this type of system. Altogether, our results can contribute towards the understanding of the endemic infection in Brazil.

Este trabalho teve por objetivo avaliar a distribuição de anticorpos neutralizantes contra o herpesvirus bovino 1 (BoHV-1) em 722 animais não vacinados pertencentes a oito propriedades que utilizam diferentes sistemas de criação (extensivo, semi-confinamento e confinamento). As amostras sorológicas foram submetidas ao teste de soroneutralização para pesquisa de anticorpos anti-BoHV-1. Os resultados foram classificados em títulos negativo, baixo, médio e alto. Uma propriedade não apresentou amostras positivas, enquanto que nas demais propriedades a frequência de amostras positivas variou de 17,95% a 86,96%. Os animais do sistema de criação por confinamento apresentaram menor número de amostras positivas quando comparado aos animais do outros sistemas de criação, possivelmente devido às boas práticas sanitárias adotadas por esse sistema. Dessa forma, nossos resultados podem contribuir para o entendimento dessa infecção endêmica no Brasil.

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

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

Aislamiento delvvirus herpes bovino tipo 1 en bovinos del departamento de córdoba - Colombia / Isoation of boviine herpes virus type 1 from cattle in cordoba - Colombia

Objetivo. Realizar el aislamiento del virus herpes bovino tipo 1 (BHV-1) en ganado bovino con antecedentes de infertilidad. Materiales y métodos. A partir de 85 animales, provenientes de diferentes áreas rurales del departamento de Córdoba, Colombia, sin antecedentes de vacunación y con titulos neutralizantes contra la enfermedad de la rinotraqueitis bovina infecciosa (IBR) por seroneutralización, se escogieron dos toros y una vaca para hacer aislamiento de HVB-1. Los animales fueron inmunosuprimidos con Dexametasona, y se obtuvieron muestras con hisopos nasales, oculares y de lavado prepucial en los toros y vaginales en la vaca respectivamente. Resultados. Se observó un efecto citopático a las 3 horas después de la inoculación de las células MDBK con el lavado genital de la vaca y a las 24 horas en los toros, con exposición del efecto en "racimo" a las 48 horas. El aislamiento en ambos tipos de muestras, sugiere que la reactivación viral fue seguida por una fase de viremia y excreción del virus en las secreciones naturales. Conclusiones. El aislamiento del virus HVB-1 en los reproductores aparentemente sanos pero con títulos a la prueba de seroneutralización, establece la presencia de latencia viral en éstos animales, importante factor epidemiológico en la difusión de la enfermedad a nivel de campo. Se deben iniciar los estudios necesarios para establecer el subtipo de virus actuante en el campo, para conocer sus características antigénicas y su correspondencia con las cepas vacunales.

Objective. Bovine herpes virus type 1 (BHV-1) was isolated from beef cattle with history of infertility. Materials and methods. We chose 85 unvaccinated animals, from different rural areas of Cordoba Department that had evidence of infectious bovine rhinotracheitis (IBR) by serum neutralization. Two bulls and one cow were chosen to attempt isolation of HVB-1. The three bovines were immunosupressed with glucocorticoids (dexamethasone) and samples of prepucial washing (males) vaginal washing (female), and nasal and ocular swaps were obtained. Results. Cytopathic effect was detected 3 hours post-inoculation in the MDBK cells inoculated with vaginal washing of the cow and 24 hours post-inoculation in the cells inoculated with washings from the bulls. A "racime effect" appeared after 48 hours. Successful isolation from both types of samples, suggests that viral reactivation was followed by viremia and shedding of virus in natural secretions and excretions. Conclusions. The isolation of BHV-1 from apparently normal reproductive animals confirms the presence of viral latency in these animals. Latency is an important epidemiological factor in the spread of the disease among farm animals. Studies should be initiated to identify the viral subtype circulating understand its antigenic characteristics and match the virus to a vaccine strain.