Avian IgY antibodies: characteristics and applications in immunodiagnostic / Anticorpos IgY aviário: características e aplicações em imunodiagnóstico

Immunoglobulin Y (IgY) is the major antibody isotype in birds, reptiles, amphibia, and lungfish, playing a similar biological role as mammal IgG. Due to its phylogenetic distance, immune diversification and presence in the egg yolk, IgY provide a number of advantages in immunodiagnostic compared to IgG from mammals. Moreover, IgY production is in agreement with international efforts to reduce, refine and if possible, to replace animals in experimentation, contributing substantially in favor of animal welfare. This article presents an overview about structural and functional features, production and applications of IgY in immunodiagnostic, as well as the advantages of chicken antibodies use.

A imunoglobulina Y (IgY) é a classe de anticorpos de maior importância em aves, répteis, anfíbios e peixes pulmonados, desempenhando um papel semelhante a IgG de mamíferos. Devido a sua distância filogenética, mecanismos de diversificação imune e presença na gema do ovo, IgY proporciona uma série de vantagens em imunodiagnóstico, quando comparada a IgG de mamíferos. Além disso, esse método alternativo de produção de anticorpo está de acordo com os esforços internacionais para reduzir, refinar e, se possível, substituir animais em experimentação, contribuindo substancialmente a favor do bem-estar animal. Este artigo apresenta uma revisão sobre as características estruturais e funcionais da IgY, bem como os métodos de produção, vantagens e aplicações em imunodiagnóstico, além das vantagens da sua utilização.

Bacterial biodiversity from an anaerobic up flow bioreactor with ANAMMOX activity inoculated with swine sludge

The present study aimed to describe the bacterial community present at an anaerobic up flow bioreactor with ANAMMOX activity, inoculated with the sludge from the anaerobic pond of a swine slurry treatment system. The description was based on the molecular DNA techniques using primers for amplification of complete 16S rRNA gene and also new primers to amplify smaller fragments from 16S rRNA. During the bioreactor operation time, the bacterial community changed significantly, increasing the nitrogen removal efficiency, reaching after 500 days a removal rate of 94 percent. The complete PCR amplification of 16S rRNA gene generated 17 clones, where three presented similarity with Candidatus Jettenia asiatica (97 percent), twelve with Janthinobacterium (99 percent) and two with uncultured clones. The PCR amplification of 436 base pairs had generated 12 clones, of which eight presented 96-100 percent similarity with Candidatus Anammoxoglobus propionicus, Planctomycete KSU-1 and one with Pseudomonas sp. (99 percent) and three with uncultured clones.

Experimental infection of rabbits with a recombinant bovine herpesvirus type 5 (BoHV-5) gI, gE and US9-negative / Infecção experimental de coelhos com um recombinante do herpesvírus bovino tipo 5 defectivo na gI, gE e US9

Bovine herpesvirus type 5 (BoHV-5) is a major cause of viral meningoencephalitis in cattle. The expression of different viral proteins has been associated with BoHV-5 neuropathogenesis. Among these, gI, gE and US9 have been considered essential for the production of neurological disease in infected animals. To evaluate the role of gI, gE and US9 in neurovirulence, a recombinant from which the respective genes were deleted (BoHV-5 gI-/gE-/US9-) was constructed and inoculated in rabbits of two age groups (four and eight weeks-old). When the recombinant virus was inoculated through the paranasal sinuses of four weeks-old rabbits, neurological disease was observed and death was the outcome in 4 out of 13 (30.7 percent) animals, whereas clinical signs and death were observed in 11/13 (84.6 percent) of rabbits infected with the parental virus. In eight weeks-old rabbits, the BoHV-5 gI-/gE-/US9- did not induce clinically apparent disease and could not be reactivated after dexamethasone administration, whereas wild type BoHV-5 caused disease in 55.5 percent of the animals and was reactivated. These findings reveal that the simultaneous deletion of gI, gE and US9 genes did reduce but did not completely abolish the neurovirulence of BoHV-5 in rabbits, indicating that other viral genes may also play a role in the induction of neurological disease.

O herpesvírus bovino tipo 5 é uma das principais causas de meningoencefalite viral em bovinos. A expressão de diferentes proteínas virais tem sido associada à neuropatogenia do BoHV-5. Entre estas, a gI, gE e US9 têm sido consideradas essenciais para a indução de sinais neurológicos nos animais infectados. Para avaliar o papel das proteínas gI, gE e US9 na neurovirulência, construiu-se um recombinante no qual os genes que codificam estas proteínas foram deletados, denominado BoHV-5 gI-/gE-/US9-. Este vírus foi inoculado em coelhos de idades diferentes (quatro e oito semanas de idade). Quando o vírus recombinante foi inoculado nos seios paranasais de coelhos de quatro semanas de idade, doença neurológica e morte foram observadas em 4 dos 13 (30,7 por cento) animais, enquanto que sinais clínicos e morte foram observados em 11/13 (84,6 por cento) dos coelhos infectados com o vírus parental. Em coelhos de oito semanas de idade, o BoHV-5 gI-/gE-/US9- não induziu sinais clínicos aparentes e, após tentativa de reativação viral por tratamento com dexametasona, o vírus não foi re-excretado. Por outro lado, o vírus selvagem causou doença clínica em 55,5 por cento dos coelhos e foi re-excretado após tratamento com dexametasona. Estes achados revelam que a deleção simultânea dos genes gI, gE e US9 reduziu mas não aboliu completamente a neurovirulência do BoHV-5 em coelhos, indicando que outros genes virais possam ter papel na indução da doença neurológica.

Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid). Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively.

A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa grandes perdas econômicas ao produtor e à agroindústria suinícola em todo o mundo. É causada pelo vírus da doença de Aujeszky (VDA), um alfaherpesvírus envelopado com genoma DNA de fita dupla e linear. O genoma do VDA codifica 11 glicoproteínas, as quais são os maiores alvos do sistema imune do hospedeiro em resposta a infecção. A glicoproteína E (gE) é uma proteína não essencial e a deleção do gene da gE é muito utilizada para a produção de vacinas com marcadores. Com o objetivo de desenvolver insumos moleculares para a produção de um teste de ELISA específico para gE do VDA, a seqüência do gene da gE foi amplificada, clonada e expressa no sistema de expressão em baculovírus. O produto da amplificação foi clonado no vetor pGem®-T Easy e subclonado no plasmídeo de expressão pFastBac™1. O DNA recombinante pFastBac-gE-VDA foi usado para a transposição sítio-específica no baculovírus recombinante (bacmid). Após seleção por antibióticos e cor, as colônias com o recombinante bacmid-pFastBac-gE-VDA foram selecionadas e a presença do gene da gE foi confirmada por PCR. O DNA recombinante viral, bacmid-pFastBac-gE-VDA, foi usado para cotransfecção de células de inseto Trichoplusia ni e a presença do recombinante e a proteína gE foi determinada por PCR, por SDS-PAGE e Western blotting, respectivamente.

Comparative pathogenicity of bovine herpesvirus 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a)

O presente estudo teve como objetivo examinar a capacidade de duas amostras de herpesvírus bovino tipo 1 (BHV-1) de diferentes subtipos (amostra EVI 123/96: BHV-1.1; amostra SV265/98: BHV-1.2a) de induzir doença respiratória em bovinos. Estas duas amostras são representativas de subtipos de BHV-1 prevalentes no Brasil. Os subtipos das amostras foram confirmados por análises com anticorpos monoclonais e com enzimas de restrição. As amostras foram inoculadas por via intranasal em sete bezerros de três meses de idade (quatro com BHV-1.1, três com BHV-1.2a), soronegativos para BHV-1, sendo outros três animais mantidos como controles não infectados. Nos dois grupos de animais inoculados, os sinais clínicos observados foram consistentes com o quadro de rinotraqueíte infecciosa bovina (IBR), incluindo febre, apatia, anorexia, descargas mucopurulentas nasais e oculares, conjuntivite, erosões e hiperemia na mucosa nasal, dispnéia, tosse, estridor traqueal e aumento dos linfonodos retrofaríngeos, submandibulares e cervicais. Não foram observadas diferenças significativas entre os escores clínicos atribuídos aos animais nos dois grupos. Igualmente, foram similares as quantidades de vírus re-isoladas dos animais infectados, à exceção de uma diferença significativa na disseminação de vírus pelas secreções nasais, a qual foi maior nos animais infectados com BHV-1.1 nos dias 1 a 3 pós-inoculação. Após reativação induzida por corticosteróides, foi observado recrudescimento dos sinais clínicos, os quais foram também similares em ambos os grupos. Em conclusão, as amostras de BHV-1 dos subtipos 1 e 2a não apresentaram diferenças significativas em sua patogenicidade sobre o trato respiratório nos animais inoculados, tanto após a infecção primária como após a reativação.

A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus

Neutralizing antibodies to bovine herpesviruses types 1 (BHV-1) and 5 (BHV-5) induced by an experimental, oil-adjuvanted, BHV-1 vaccine

An experimental oil-adjuvanted, inactivated vaccine against bovine herpesvirus type 1 (BHV-1.1), was produced and evaluated in its capacity to induce neutralizing antibodies against bovine herpesvirus type 1 (BHV-1, subtypes 1.1 and 1.2) and bovine herpesvirus type 5 (BHV-5). Cattle were vaccinated and revaccinated 90 days later. Antibodies were measured at days 0, 30, 90, 120, 180, 270 and 450 days after the first dose of vaccine (DPV). Antibody titres to BHV-1.1 and BHV-1.2 were significantly higher than to BHV-5 throughout the experiment. While all calves seroconverted to BHV-1.1 and BHV-1.2 after the first dose of vaccine, only two out of 23 (8,7 percent) calves seroconverted to BHV-5. However, after the booster injection all animals seroconverted to the three virus types. At 450 DPV, 79 percent (15/19 cattle) and 84 percent (16/19) were still positive for antibodies to BHV 1.1 and BHV 1.2, whereas 50 percent (10/19) of the calves remained seropositive for BHV-5. It was concluded that although a potent BHV-1 vaccine may induce crossreactive neutralizing antibodies to BHV-5, the levels of such antibodies are significantly lower and of shorter duration than antibodies to BHV-1.1 or BHV-1.2