RESUMO
As neoplastic viruses have been affecting Iranian chicken farms more frequently in recent years, the first step in prevention may therefore be to genetically characterize and systematically identify their source and origin. Recently, we published a phylogenetic analysis based on the meq gene of Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1), that circulated in Iranian backyard and commercial chickens. In the current study, we are reporting for the first time the identification of a 298 aa meq protein containing only two PPPP motifs from an MDV-1-infected unvaccinated backyard turkey. This protein length has never been reported from any turkey species before. According to phylogenetic analysis, a close genetic relationship (0.68%) to several chicken-origin isolates such as the American vv + 648A strain was found. In addition, we identified a standard meq protein from a MDV-1-infected commercial chicken farm. In corroboration with our previous finding from other Iranian provinces, it is likely that the highly identical MDV-1 viruses currently circulating in Iranian chicken farms, which may be indicative of human role in the spread of the virus, have similar Eurasian origin. Our data suggest that regardless of the meq size, MDV-1 circulating in Iran are from different origins. On the other hand, meq sequences from bird species other than chicken have been reported but are very few. Our investigation suggests MDV-1 circulating in turkey do not have species-specific sequences.
Assuntos
Herpesvirus Galináceo 2 , Doença de Marek , Doenças das Aves Domésticas , Animais , Galinhas , Herpesvirus Galináceo 2/genética , Humanos , Irã (Geográfico)/epidemiologia , Doença de Marek/epidemiologia , Doença de Marek/prevenção & controle , Filogenia , Aves Domésticas , Doenças das Aves Domésticas/epidemiologiaRESUMO
In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45-UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.
Assuntos
Herpesvirus Galináceo 2 , Doença de Newcastle , Doenças das Aves Domésticas , Vacinas Virais , Animais , Anticorpos Antivirais , Sistemas CRISPR-Cas , Galinhas , Genótipo , Herpesvirus Meleagrídeo 1/genética , Integrases , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/genética , Vacinas Sintéticas/genética , Vacinas Virais/genéticaRESUMO
Background: Marek's disease (MD) is a transmissible disease in chickens caused by Gallid alphaherpesvirus 2 (GaHV-2). The infection is characterized by lymphocyte cellular infiltrates in peripheral nerves and other organs and tissues, including the skin; which can lead to dysfunction causing progressive asymmetric paresis and complete spastic paralysis of body extremities. Dermatitis and cardiac myositis caused by GaHV-2 in free-range chickens has rarely been described in Brazil. This reports the occurrence of the disease with a confirmatory molecular diagnosis in free-range poultry showing signs of dermatitis, poor performance, and cachexia and no mortality in the semi-arid Potiguar region. Cases: Twenty roosters of the Shamo lineage, among a brood of 42 birds, had a history of progressive weight loss and skin lesions. Two birds with poor body condition, erythema, and scaling of the skin in the head and cervical regions were sent for clinical care. All birds were between 12 and 18 months of age and were vaccinated against Newcastle disease and Fowlpox with only a few receiving vaccines against MD and Gumboro disease. According to the owner's report, some birds were previously kept outdoors, and when they were transferred to a small shed with little air circulation, they began to develop clinical signs after approximately 15 days. The first signs of the disease were also reported to have appeared 2.5 months before clinical care and, in the meantime, several treatments were instituted without success. Owing to the general condition of the animals and inconclusive clinical suspicion, the birds were subjected to euthanasia and necropsy. Tissue samples were collected for histopathological and polymerase chain reaction analyses to search for the GaHV-2 DNA meq gene. The main clinicopathological findings were erythema (47%, 20/42) and desquamation of skin and mild, prominent white multifocal areas in the heart. Histopathology revealed infiltration of pleomorphic lymphoblastic cells in the skin, heart, and sciatic nerve. The amplification of the L-meq and meq oncoprotein genes in these organs and in the liver, confirmed the infection by GaHV-2, consistent with that of a field strain. Discussion: MD was confirmed based on the macroscopic and histological lesions, and with the detection of GaHV-2 DNA in the affected tissues. The unusual clinical presentation represented an initial challenge for diagnosis. The clinical history was important to lead to the suspicion of MD, as roosters initiated clinical signs 15 days after they were transferred to a small shed with poor air circulation. This probably favored the high viral concentration and disease transmission among susceptible birds in the brood because the feather follicle is the primary site of viral replication for transmission; and desquamation of infected epithelial cells favor airborne horizontal transmission to susceptible chickens. The roosters had not been vaccinated against MD, which probably favored the infection, as vaccination is known to be a fundamental approach for MD control for effective growth of the poultry industry. Clinical findings and lesions, together with viral molecular detection, were fundamental for the diagnosis, a premise for the application of adequate prevention and control measures for the disease in breeding. This is the first report of MD with a confirmatory molecular diagnosis in northeastern Brazil.
Assuntos
Animais , Masculino , Galinhas/virologia , Doença de Marek/diagnóstico , Herpesvirus Galináceo 2/isolamento & purificação , Proto-Oncogenes , Reação em Cadeia da Polimerase/veterinária , Dermatite/veterinária , Miosite/veterináriaRESUMO
Marek's disease virus (MDV) is an immunosuppressive pathogen that can cause low production efficiency and high mortality rates in chickens. There is no current information on the MDV serotypes and pathotypes circulating in vaccinated commercial farms in Colombia where the birds are vaccinated in the incubator with Gallid herpesvirus (GaHV-2) and Meleagrid herpesvirus 1 (MeHV-1). Based on that, the main focus of this study was to understand the MDV's infection dynamics for the three known serotypes and to detect wild-virus pathogenic strains in 4-layer poultry farms in Antioquia. Samples of blood, feathers and spleens were collected from three randomly chosen animals according to age category: 1, 15, 30, 60, 90, and 120 days. Quantitative real-time PCR (qPCR) that differentiates between the three serotypes of MDV was used to assess viral loads over time, and phylogenetic analysis of the Meq oncogene was done to compare the strains of MDV with those of known pathogenicity. Meleagrid herpesvirus 1 (MeHV-1) was detected in all blood and feather follicle samples with an average number of genome copies (per 10,000 cells) of 31.44 in blood as expected as a result of vaccination. GaHV-2 was also detected in almost 100% of the blood and feather follicle samples throughout all defined age categories, with an average of 10.65 genome copies in blood samples. Gallid herpesvirus 3 (GaHV-3) was detected in 72% of blood and 84.61% of feather samples, with less than 1 copy per 10,000 cells. Based on the number of 132 bp repeats of the BamHI-H and BamHI-D regions in pooled feather samples, there were 70% (8/25) of attenuated MDV and 30% (17/25) of virulent MDV strains circulating in the farms. Virus isolation was performed successfully from every farm. In conclusion, different strains of MDV are circulating for up to 120 days in layers in Antioquia-Colombia and could be of major impact in poultry health. Keywords: Marek's disease virus (MDV); Antioquia-Colombia; qPCR; PCR; Meq gene phylogeny.
Assuntos
Galinhas , Plumas , Herpesvirus Galináceo 2 , Doença de Marek , Animais , Galinhas/virologia , Colômbia , Plumas/virologia , Feminino , Herpesvirus Galináceo 2/genética , Doença de Marek/diagnóstico , Doença de Marek/virologia , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologiaRESUMO
In India, increasing incidence of Mareks Disease Virus (MDV) outbreaks are being reported even in vaccinated poultry farms. Hence identifying the new emerging pathotype of MDV is necessary for successful control through vaccination. Birds received in the post mortem section of The Avian Disease Laboratory, Indian Veterinary Research Institute, were screened for the presence of MDV by collecting neoplastic tissues, spleen and feather follicles. Screening was carried out by polymerase chain reaction (PCR) and histopathological examination. Among the tested 150 birds tissue samples, 35 bird tissue samples were found positive for MDV. Based on pathotyping specific PCR, it was found that 34 birds tissues were affected virulent MDV and one birds tissue was affected with very virulent MDV. Since, HVT vaccine will not protect the very virulent pathotype, combined vaccine of SB-1 and HVT can be administered to control the very virulent MDV. Among the MD infected birds, neoplastic liver is most commonly encountered. Spleen tissue samples was found to be more suitable for the DNA isolation for PCR.(AU)
Assuntos
Animais , Doença de Marek/diagnóstico , Doença de Marek/patologia , Herpesvirus Galináceo 2 , Reação em Cadeia da Polimerase/veterinária , Aves Domésticas , ÍndiaRESUMO
Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.
Assuntos
Galinhas/virologia , Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 2/isolamento & purificação , Doença de Marek/virologia , Doenças das Aves Domésticas/virologia , Animais , Células Cultivadas , Embrião de Galinha , Colômbia , DNA Viral , Feminino , Fibroblastos/virologia , Herpesvirus Galináceo 2/classificação , Herpesvirus Galináceo 2/patogenicidade , Doença de Marek/patologia , Filogenia , Doenças das Aves Domésticas/patologia , Neuropatia Ciática/veterinária , Neuropatia Ciática/virologia , Análise de Sequência de DNA , Análise de Sequência de Proteína , Sorotipagem , Timo/virologiaRESUMO
Marek's disease (MD) is a lymphoproliferative disorder caused by Gallid herpesvirus 2 (MDV) that infects mainly domestic gallinaceous birds although wild birds may occasionally be affected. The current report describes the anatomopathological and molecular findings of a case of MD in a white-peafowl (Pavo cristatus). The signs included apathy, hyporexia, and diarrhea. Grossly, 0.5 to 1.5cm in diameter, yellow, soft nodules were observed in the skeletal muscle, lung, kidney, air sacs, small intestine, heart, ovary, ventriculus, and proventriculus. Microscopically, numerous atypical round neoplastic cells were noted. The molecular detection of MDV DNA was implemented to amplify part of the meq gene and products were sequenced for the phylogenetic analysis. Template DNA was obtained from tissues of the affected bird and from blood of all the gallinaceous birds of the Zoo. The expected amplicon for the partial amplification of MDV meq gene was obtained and the amplicons were sequenced. Sequences obtained enabled grouping the strain (accession no. KT768121) with MDV serotype 1 strains from the GenBank. Based on the anatomopathological and molecular findings, the diagnosis of MD in a white-peafowl was reached, and to the authors' knowledge, no previous report regarding MD was published in Pavo cristatus.(AU)
Doença de Marek (MD) é uma desordem linfoproliferativa causada pelo Gallid herpesvirus 2 (MDV), que infecta principalmente galináceos domésticos, porém aves silvestres podem ser ocasionalmente afetadas. O presente relato descreve os achados anatomopatológicos e moleculares de um caso de MD em um pavão-branco (Pavo cristatus). Os sinais clínicos incluíram apatia, hiporexia e diarreia. Macroscopicamente, foram observados nódulos macios, de 0,5 a 1,5cm de diâmetro, no músculo esquelético, no pulmão, nos rins, nos sacos aéreos, no intestino delgado, no coração, no ovário, no ventrículo e no proventrículo. Microscopicamente, numerosas células redondas neoplásicas atípicas foram notadas. A detecção molecular do DNA do MDV foi implementada para amplificar parte do gene meq, e os produtos foram sequenciados para análise filogenética. DNA foi obtido de tecidos de aves afetadas e do sangue de todos os galináceos do zoológico. A esperada amplificação de parte do gene meq de MDV amplificado foi ampliada e sequenciada. As sequências obtidas permitiram o agrupamento da cepa (acesso KT768121) com cepas do sorotipo 1 de MDV do GenBank.. O diagnóstico de MD em pavão-branco foi obtido com base nos achados anatomopatológicos e moleculares e, pelo conhecimento dos autores, não há relatos anteriores publicados de MD em Pavo cristatus.(AU)
Assuntos
Animais , Galliformes/virologia , Herpesvirus Galináceo 2/isolamento & purificação , Doença de Marek/diagnóstico , Linfoma/veterinária , Vírus OncogênicosRESUMO
Marek's disease (MD) is a lymphoproliferative disorder caused by Gallid herpesvirus 2 (MDV) that infects mainly domestic gallinaceous birds although wild birds may occasionally be affected. The current report describes the anatomopathological and molecular findings of a case of MD in a white-peafowl (Pavo cristatus). The signs included apathy, hyporexia, and diarrhea. Grossly, 0.5 to 1.5cm in diameter, yellow, soft nodules were observed in the skeletal muscle, lung, kidney, air sacs, small intestine, heart, ovary, ventriculus, and proventriculus. Microscopically, numerous atypical round neoplastic cells were noted. The molecular detection of MDV DNA was implemented to amplify part of the meq gene and products were sequenced for the phylogenetic analysis. Template DNA was obtained from tissues of the affected bird and from blood of all the gallinaceous birds of the Zoo. The expected amplicon for the partial amplification of MDV meq gene was obtained and the amplicons were sequenced. Sequences obtained enabled grouping the strain (accession no. KT768121) with MDV serotype 1 strains from the GenBank. Based on the anatomopathological and molecular findings, the diagnosis of MD in a white-peafowl was reached, and to the authors' knowledge, no previous report regarding MD was published in Pavo cristatus.(AU)
Doença de Marek (MD) é uma desordem linfoproliferativa causada pelo Gallid herpesvirus 2 (MDV), que infecta principalmente galináceos domésticos, porém aves silvestres podem ser ocasionalmente afetadas. O presente relato descreve os achados anatomopatológicos e moleculares de um caso de MD em um pavão-branco (Pavo cristatus). Os sinais clínicos incluíram apatia, hiporexia e diarreia. Macroscopicamente, foram observados nódulos macios, de 0,5 a 1,5cm de diâmetro, no músculo esquelético, no pulmão, nos rins, nos sacos aéreos, no intestino delgado, no coração, no ovário, no ventrículo e no proventrículo. Microscopicamente, numerosas células redondas neoplásicas atípicas foram notadas. A detecção molecular do DNA do MDV foi implementada para amplificar parte do gene meq, e os produtos foram sequenciados para análise filogenética. DNA foi obtido de tecidos de aves afetadas e do sangue de todos os galináceos do zoológico. A esperada amplificação de parte do gene meq de MDV amplificado foi ampliada e sequenciada. As sequências obtidas permitiram o agrupamento da cepa (acesso KT768121) com cepas do sorotipo 1 de MDV do GenBank.. O diagnóstico de MD em pavão-branco foi obtido com base nos achados anatomopatológicos e moleculares e, pelo conhecimento dos autores, não há relatos anteriores publicados de MD em Pavo cristatus.(AU)
Assuntos
Animais , Doença de Marek/diagnóstico , Herpesvirus Galináceo 2/isolamento & purificação , Galliformes/virologia , Linfoma/veterinária , Vírus OncogênicosRESUMO
Isolates of Marek's disease virus (MDV) from vaccinated flocks in Argentina were characterized as very virulent (vv) and very virulent plus (vv+) strains. Experimental infection with these viruses caused a high incidence of Marek's disease in both resistant N-2a line and susceptible P-2a line birds. Vaccine viruses from each of the three Marek's disease viral serotypes were evaluated alone and in various combinations for protection against challenge with a vvMDV called NULP-1. Vaccination of P-2a birds with HVT did not protect satisfactorily against any of the vv and vv+MDV strains isolated. However, CVI988/Rispens vaccine alone or combined with serotype 2 and/or serotype 3 vaccine strains enhanced protection significantly against NULP-1. Serotype 2 plus serotype 3 vaccines also provided significant protection when challenged with this strain. This is one the first reports of the occurrence of vvMDV and vv+MDV in Argentina and Latin America. It is also a preliminary evaluation of the synergistic protective effect of different vaccine viruses with local MDV strains. However, further studies are needed to evaluate the real role of these and other Marek's disease isolates in 'vaccination failures' and the influence of serotype and virus strain on synergism between Marek's disease vaccine viruses.
Assuntos
Herpesvirus Galináceo 2/classificação , Herpesvirus Galináceo 2/patogenicidade , Vacinas contra Doença de Marek/imunologia , Doença de Marek/virologia , Doenças das Aves Domésticas/virologia , Animais , Argentina/epidemiologia , Galinhas/virologia , Predisposição Genética para Doença , Doença de Marek/epidemiologia , Doença de Marek/imunologia , Doença de Marek/prevenção & controle , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Organismos Livres de Patógenos Específicos , VirulênciaRESUMO
Amostras de sangue foram obtidas ao abate de 60 frangos de corte, de 43 a 56 dias de idade, de cada uma de 60 granjas sem problemas sanitários aparentes localizadas em uma regiäo de alta densidade de produçäo avícola. As granjas correspondiam a 10% do número total que existe na regiäo e estavam localizadas em um raio de 50 km. Todos os frangos tinham sido vacinados contra a doença de Marek no primeiro dia de vida. Os soros foram avaliados no teste de imunodifusäo para anticorpos para reovírus aviário (RVA), vírus da doença infecciosa da bursa (VDIB), vírus da bouba aviária (VBA), vírus herpes de perus (HVT), adenovírus aviário do grupo 1 (AVA-1) e do grupo 2 (AVA-2). Os mesmos soros foram examinados no microteste de soroneutralizaçäo em placas para anticorpos para o vírus da bronquite infecciosa (VBI) e o vírus da laringotraqueíte infecciosa (VLTI), bem como anticorpos inibidores da hemoaglutinaçäo para o vírus da doença de Newcattle (VDN). Anticorpos para RVA, detectados em 2172 (63,6%) de 3418 soros, e para AVA-1, em 2701 (78,2%) de 3456 soros, foram demonstrados em todas as 60 granjas, enquanto que, anticorpos para o VDIB, em 3086 (89,4%) de 3452 soros, foram encontrados em somente 57 destas granjas. Estes resultados indicam que os três vírus säo ubíquos em frangos de corte da regiäo estudada. Anticorpos para o VBA em 14 (0,5%) de 2935 soros, para o VDN em seis (0,2%) de 3320 soros, foram encontrados em cinco de 59 e em uma de 60 granjas, respectivamente, indicando que esses dois vírus ocorrem apenas raramente. Anticorpos para HVT, em 315 (9,0%) de 3484 soros, foram detectados em 39 de 60 granjas testadas, sugerindo resposta sorológica pobre à vacinaçäo e baixa exposiçäo de campo ao vírus da doença de Marek. Anticorpos para o VBI, em 61 (2,1%) de 2925 soros, foram encontrados em 12 de 57 granjas testadas. Porém, em apenas uma granja a percentagem de reagentes era significativa (41%); nas outras 11 granjas a percentagem de frangos com anticorpos variava de 1,7 a 16,1%. Finalmente, näo foram detectados anticorpos em 3491 soros de 60 granjas testada para AVA-2, nem em 3035 soros de 59 granjas testadas para o VLTI, indicando que esses vírus näo existem na área geográfica estudada. O significado de todos os achados é discutido