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1.
Front Microbiol ; 13: 1042212, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36532420

RESUMO

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae.

2.
Arch Virol ; 167(4): 1151-1155, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35244762

RESUMO

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.


Assuntos
Infecções por Herpesviridae , Herpesvirus Galináceo 1 , Doenças das Aves Domésticas , Animais , Galinhas , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA
3.
PLoS One ; 16(12): e0261122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34914770

RESUMO

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn't compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.


Assuntos
Membrana Corioalantoide/virologia , Vírus da Varíola das Aves Domésticas/isolamento & purificação , Varíola Aviária/diagnóstico , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pele/virologia , Sequenciamento Completo do Genoma/métodos , Animais , Austrália , Embrião de Galinha , Galinhas , Varíola Aviária/virologia , Vírus da Varíola das Aves Domésticas/classificação , Vírus da Varíola das Aves Domésticas/genética , Vírus da Varíola das Aves Domésticas/crescimento & desenvolvimento , Polimorfismo Genético
4.
Infect Genet Evol ; 96: 105095, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34597819

RESUMO

Avian hepatitis E virus (aHEV) is the causative agent of an important disease of broiler breeders and layers. aHEV cannot be readily propagated in cell culture and is characterised primarily by sequencing of amplicons generated through several RT-PCRs that target individual genes. This study aims to uncover the origin of current Australian aHEV isolates based on whole genome sequencing using clinical liver tissues. Complete genome sequences of the two aHEV isolates were assembled using Nanopore and Illumina reads. The two isolates possessed only four single nucleotide polymorphisms to each other. Comparison of the sequences with aHEV genome sequences available in the GenBank showed the highest nucleotide sequence identity of 88% with the prototype USA strain (AY535004), 82% with the European (AM943647) and genotype 1 Australian strains (AM943647). Recombination analysis suggested that aHEV isolates characterised in this study are progeny of a cross between a US and a Hungarian strain. Phylogenetic tree and phylogenetic networks constructed using complete genome and individual coding sequences revealed that Australian aHEV isolates formed a distinct clade closer to the USA strains and classified as genotype 2 whereas genotype 1 Australian strain clustered together with South Korean strains.


Assuntos
Galinhas , Genoma Viral , Hepatite Viral Animal/virologia , Hepevirus/genética , Doenças das Aves Domésticas/virologia , Infecções por Vírus de RNA/veterinária , Animais , Feminino , Fígado/virologia , Filogenia , Infecções por Vírus de RNA/virologia , Recombinação Genética , Sequenciamento Completo do Genoma
5.
J Virol Methods ; 283: 113907, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32502499

RESUMO

Characterisation of the entire genome of Fowl aviadenoviruses (FAdV) requires isolation and propagation of the virus in chicken embryo liver or kidney cells, a process which is not only time consuming but may occasionally fail to result in viral growth. Furthermore, in a mixed infection, isolation in cell culture may result in the loss of viral strains. In this study, we optimised a FAdV DNA extraction technique directly from affected liver tissues using kaolin hydrated aluminium silicate treatment. The whole genome of FAdV was sequenced directly from extracted DNA without any targetted PCR based enrichment. The extraction method was also tested on avian liver tissues affected with the RNA virus Avian hepatitis E virus and demonstrated to yield sequencing grade RNA. Therefore, the method described here is a simple technique which is potentially useful for the extraction of sequencing grade DNA/RNA from tissues with high fat content.


Assuntos
Aviadenovirus/genética , DNA Viral/isolamento & purificação , Fígado/virologia , RNA Viral/isolamento & purificação , Sequenciamento Completo do Genoma/métodos , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/isolamento & purificação , Galinhas/virologia , Genoma Viral , Hepatite , Hepevirus/genética , Sequenciamento de Nucleotídeos em Larga Escala
6.
Infect Genet Evol ; 78: 104067, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31678646

RESUMO

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract illness and substantial economic losses to the commercial poultry industry worldwide. Due to its geographical isolation, Australia has had a unique population of ILTV genotypes, and this has provided the researchers with an excellent opportunity to examine the evolution of herpesviruses. Recent studies on the evolution of ILTV have reported the emergence of recombinant ILTVs in Australian poultry flocks. More recently, there has been an increasing number of field outbreaks caused by ILTV isolates that are indistinguishable from serva vaccine strain using current molecular tests that rely on restriction fragment analysis of selected regions of the viral genome. In this study, whole-genome analysis of one of the field isolates revealed a new class of ILTV, identified here as class 7b, emerged as a result of recombination probably between another recombinant strain and the Serva vaccine strain (now reclassified as 7a). Interestingly, the 7b virus had the highest similarity to class 9, a virus that dominates the ILTV population in Victoria, where 7b has never been reported to date. Also, sequence analysis detected sequences unique to class 10, another recombinant virus that became predominant in some states of Australia between 2013 and 2014 but disappeared since then. These results demonstrate the influence of recombination as a continuous process towards more virulent and transmissible ILTVs.


Assuntos
Infecções por Herpesviridae/virologia , Herpesvirus Galináceo 1/classificação , Vírus Reordenados/genética , Vacinas Virais/genética , Sequenciamento Completo do Genoma/métodos , Animais , Austrália , Galinhas/virologia , Genoma Viral , Genótipo , Herpesvirus Galináceo 1/genética , Herpesvirus Galináceo 1/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Recombinação Genética
7.
Avian Pathol ; 48(5): 396-405, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31042049

RESUMO

Fowl aviadenoviruses (FAdV) are important avian pathogens, responsible for several poultry diseases prevalent worldwide, including inclusion body hepatitis (IBH). FAdV intraspecies cross-protection has been clearly demonstrated, but there is little evidence that any interspecies cross-protection exists. The present study aimed to assess the inter- and intraspecies protection between three FAdV field isolates (FAdV-8a, FAdV-8b, FAdV-11) identified in association with severe IBH outbreaks. Inocula prepared using inactivated plaque-purified virus with adjuvant Montanide™ ISA 71VG, were injected intramuscularly into 3-week-old SPF chickens. At 6-weeks of age, the birds were challenged with 106 TCID50 of homologous or heterologous virus intraperitoneally, and full post mortem examination performed at 4 days post-challenge. Various tissues were examined for gross and histological lesions and assessed for the presence of virus by PCR-HRM. All homologous-type vaccine/challenge groups exhibited protection against IBH lesions with no virus detected in the tissues. Unvaccinated groups challenged with virus showed evidence of FAdV-induced lesions; however, FAdV-8a demonstrated lower pathogenicity compared with FAdV-8b and FAdV-11. In the heterologous-type vaccine/challenge groups, FAdV-8a vaccine was shown to protect against challenge with both FAdV-8b and FAdV-11. FAdV-8a and 8b belong to species E and were therefore anticipated to cross-protect. However, FAdV-11 belongs to species D and therefore cross-protection by FAdV-8a was an uncharacteristic and unique finding of this study. Further research is required to disseminate the molecular basis for the interspecies cross-protection between FAdV-8a and FAdV-11. Nonetheless, the FAdV-8a isolate was shown to have substantial potential as a vaccine candidate in countries where FAdV-8a, 8b or 11 are prevalent.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/imunologia , Galinhas/imunologia , Hepatite Viral Animal/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Vacinação/veterinária , Vacinas Virais/imunologia , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/virologia , Animais , Galinhas/virologia , Proteção Cruzada , Hepatite Viral Animal/virologia , Corpos de Inclusão , Doenças das Aves Domésticas/virologia , Sorogrupo , Especificidade da Espécie , Organismos Livres de Patógenos Específicos
8.
Vet Microbiol ; 231: 48-55, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955823

RESUMO

Mycoplasma synoviae (MS) is a major pathogen of poultry globally, causing chronic respiratory disease and arthritis. Vaccination is an effective means for the control of the disease. The MS-H vaccine is an attenuated strain developed through chemical mutagenesis of an Australian field strain, 86079/7NS. Analysis of whole genome of MS-H and its comparison with that of 86079/7NS has revealed a frameshift mutation early in a gene (oppF) that codes for an oligopeptide transporter permease, OppF. Monospecific antibodies raised against peptides upstream and downstream of the mutation in OppF revealed that only N-terminus of the OppF was expressed in MS-H while the full version was expressed in 86079/7NS. Also, examination of the recombinant N- (OppF-N) and C termini (OppF-C) of OppF, upstream and downstream of the mutation site respectively, as well as the full length OppF in Western immunoblotting experiments showed that serum from MS-H vaccinated chicken strongly bound OppF-N while serum from 86079/7NS challenged chicken detected OppF, OppF-N and OppF-C. The potential of the recombinant OppF, OppF-N and OppF-C to discriminate antibody responses to MS-H reisolates with wild or vaccine type OppF was assessed against 88 chicken sera in indirect ELISA and ratios were calculated between optical densities (OD) over those obtained in MS major membrane protein MSPB ELISA. Comparison of the OD ratios revealed that the MSPB/OppF and MSPB/OppF-C OD ratios of the sera against isolates with vaccine type OppF were significantly higher than those against isolates with wild type OppF. These results are in accordance with oppF gene mutation in MS-H and confirms that MS-H does not express OppF beyond the frame shift mutation found in its oppF gene. Also, the indirect ELISA based on OppF-C in combination with the MSPB has the potential to differentiate between MS-H and field strain antibody responses.


Assuntos
Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Mutação , Infecções por Mycoplasma/veterinária , Mycoplasma synoviae/genética , Doenças das Aves Domésticas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Austrália , Proteínas de Bactérias/imunologia , Galinhas , Ensaio de Imunoadsorção Enzimática , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/prevenção & controle , Mycoplasma synoviae/imunologia , Doenças das Aves Domésticas/prevenção & controle , Testes Sorológicos , Vacinas Atenuadas/imunologia
9.
Vet Microbiol ; 228: 252-258, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593375

RESUMO

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes upper respiratory tract disease in chickens and significant losses to the poultry industry worldwide. Both antibody and cell-mediated responses are generated against ILTV infection; however, the correlation of humoral immune response with protection against ILTV infection is debatable. To examine if whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, four ILTV glycoproteins (gD, gE, gG and gJ) were expressed as recombinant proteins and used in conjunction with commercially available recombinant gC and gI in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-ILTV vaccination sera found that gE was the most antigenic glycoprotein and that gC ODs were strongly correlated with those of gI and gJ, while ODs to gG had a relatively poor correlation with those of other glycoproteins. Moderate to poor correlations were found between microscopic tracheal lesion scores and ODs to individual glycoproteins. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation with protective immunity as measured by the severity of clinical signs, gross lesions, and tracheal viral load. Results from this study demonstrated that systemic antibody titers to individual ILTV glycoproteins C, D, E, G, I and J had a relatively poor correlation to protective immunity.


Assuntos
Anticorpos Antivirais/sangue , Galinhas/imunologia , Herpesvirus Galináceo 1/imunologia , Glicoproteínas de Membrana/imunologia , Doenças das Aves Domésticas/imunologia , Vacinas Virais/imunologia , Animais , Galinhas/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Células HEK293 , Humanos , Imunidade Humoral , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Doenças das Aves Domésticas/virologia , Vacinação/veterinária , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificação
10.
Avian Dis ; 61(4): 512-519, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29337616

RESUMO

Inclusion body hepatitis (IBH) is a disease affecting broiler chicken flocks worldwide. Several serotypes of fowl adenovirus (FAdV) have been implicated in disease outbreaks, with and without immunosuppression as a predisposing factor. IBH usually occurs in flocks up to 30 days of age; it is seldom seen in older birds. The objective of this study was to determine whether the pathogenicity for older birds of three FAdV field strains, belonging to serotypes 1, 8b, and 11, in the absence of immunosuppressive factors, was akin to that for younger birds, and to establish an effective and economical disease model for assessing cross-protection between serotypes. To achieve this objective, the gross pathology, histopathology, and dissemination of virus were examined at multiple time points after inoculation of 6-wk-old, specific-pathogen-free chickens via intraperitoneal injection. Both FAdV-8b and FAdV-11 generated lesions typical of those associated with outbreaks of IBH, and they were shown to be primary pathogens. The presence and severity of hepatic lesions were used to define two disease stages: degeneration (1-5 days postinoculation) and convalescence (6-14 days postinoculation). During the degenerative stage, FAdV-8b was detected in the liver, kidney, and gizzard of most birds, whereas FAdV-11 was predominantly detected in the liver, and both viruses persisted in the gizzard into convalescence. The pathogenesis of two IBH-associated FAdV strains in 6-wk-old chickens confirms their high level of virulence and also provides an effective experimental model for investigation of cross-protection between FAdVs. It also demonstrates persistence of the virus in the gizzard long after infection, supporting the notion that it is a site of viral shedding.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/imunologia , Galinhas , Doenças das Aves Domésticas/prevenção & controle , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/virologia , Animais , Proteção Cruzada , Adenovirus A das Aves/imunologia , Doenças das Aves Domésticas/virologia , Sorogrupo , Organismos Livres de Patógenos Específicos , Vacinas Virais/imunologia , Virulência , Eliminação de Partículas Virais
11.
Infect Genet Evol ; 43: 67-73, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27223632

RESUMO

Infectious laryngotracheitis (ILT) is a significant viral disease of chickens in many countries around the globe. In this report the status of ILT in Australia has been used as a model to evaluate the evolution of the ILT viruses (ILTVs). Due to its geographical isolation, Australia harbored a distinct lineage of ILT viruses (ILTV) up to 2007. However examination of the ILT viruses (ILTV) involved in outbreaks between 2007 and 2009 has revealed that many of the outbreaks were caused by two new viral genotypes, class 8 and class 9. These two recombinant viruses were found to emerge as a result of recombination between previously existing live vaccine strains (SA2 and A20), and another live vaccine strain (Serva) introduced into the country in 2007. The new recombinant ILTVs were also shown to possess significantly higher virulence and replication capacity compared with a previously predominant ILTV, class 2. In the current study, examination of a large number of ILTVs isolated from outbreaks between 2009 and 2015 revealed the emergence of yet another recombinant virus (class 10) that appears to have become a predominant genotype in New South Wales. In Victoria however, the recombinant class 9 gradually became the predominant virus, replacing class 2. Therefore, there was an unusual pattern in geographical spread of the newly emerged viruses in different states of the country. These results suggest that ILTV is fast evolving towards a greater transmissibility and therefore greater capacity to spread into ILTV-free areas.


Assuntos
Galinhas/virologia , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/classificação , Doenças das Aves Domésticas/virologia , Animais , Austrália/epidemiologia , Transferência Genética Horizontal , Infecções por Herpesviridae/virologia , Herpesvirus Galináceo 1/genética , Herpesvirus Galináceo 1/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Filogenia , Doenças das Aves Domésticas/epidemiologia , Análise de Sequência de DNA/veterinária , Virulência , Replicação Viral
12.
Avian Pathol ; 44(2): 106-13, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25609454

RESUMO

Fowl adenoviruses (FAdVs) cause diseases in domestic chickens, including inclusion body hepatitis (IBH), with immunosuppression believed to play a role in their pathogenesis. To gain a better understanding of the pathogenesis and chronology of disease caused by FAdVs, the gross pathology, histopathology and dissemination of virus were examined at several different time points, after inoculation of one-day-old specific pathogen-free chickens with FAdV-1, FAdV-8b or FAdV-11 via the ocular route. FAdV-8b had a slightly greater virulence than FAdV-11, but both were primary pathogens. The presence and severity of hepatic lesions were used to define the three stages of the disease: incubation (1-3 days post-inoculation, PI), degeneration (4-7 days PI) and convalescence (14 days PI). Both viruses were detected in the liver, kidney, bursa, thymus and gizzard of most birds during the degenerative stage, and persisted in the gizzard into convalescence. The FAdV-1 isolate was found to be apathogenic, but virus was detected in the bursa and/or gizzard of several birds between 2 and 7 days PI. This is the first study examining the chronology of gross and microscopic lesions of pathogenic and apathogenic FAdVs in association with viral presence in multiple tissues. It was concluded that both FAdV-8b and FAdV-11 are primary pathogens, and that these strains may play a role in immunosuppression.


Assuntos
Infecções por Adenoviridae/veterinária , Animais Recém-Nascidos/virologia , Aviadenovirus/patogenicidade , Galinhas , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/patologia , Fatores Etários , Animais , Aviadenovirus/isolamento & purificação , Técnicas Histológicas/veterinária , Razão de Chances , Sorogrupo , Especificidade da Espécie , Organismos Livres de Patógenos Específicos , Virulência , Vísceras/patologia , Vísceras/virologia
13.
Vaccine ; 30(50): 7193-8, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-23084851

RESUMO

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated vaccines. Glycoprotein G (gG) is a virulence factor in ILTV and a gG deficient strain of ILTV (ΔgG-ILTV) has shown potential for use as a vaccine. In the poultry industry vaccination via drinking water is common, but technology is now available to allow quicker and more accurate in ovo vaccination of embryos at 18 days of incubation. In this study ΔgG-ILTV was delivered to chicken embryos at three different doses (10(2), 10(3) and 10(4) plaque forming units per egg) using manual in ovo vaccination. At 20 days after hatching, birds were challenged intra-tracheally with wild type ILTV and protection was measured. In ovo vaccination was shown to be safe, as there were no developmental differences between birds from hatching up to 20 days of age, as measured by weight gain. The highest dose of vaccine was the most efficacious, resulting in a weight gain not significantly different from unvaccinated/unchallenged birds seven days after challenge. In contrast, birds vaccinated with the lowest dose showed weight gains not significantly different from unvaccinated/challenged birds. Gross pathology and histopathology of the trachea reflected these observations, with birds vaccinated with the highest dose having less severe lesions. However, qPCR results suggested the vaccine did not prevent the challenge virus replicating in the trachea. This study is the first to assess in ovo delivery of a live attenuated ILTV vaccine and shows that in ovo vaccination with ΔgG-ILTV can be both safe and efficacious.


Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/imunologia , Óvulo/virologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Vacinas Virais/imunologia , Animais , Galinhas , Deleção de Genes , Glicoproteínas/genética , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Herpesvirus Galináceo 1/genética , Herpesvirus Galináceo 1/patogenicidade , Doenças das Aves Domésticas/patologia , Traqueia/patologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/genética , Fatores de Virulência/genética
14.
Avian Dis ; 56(3): 509-15, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23050467

RESUMO

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction with the gG-ve ILTV vaccine in a 'differentiating infected from vaccinated animals' approach to the control of ILT.


Assuntos
Anticorpos Antivirais/sangue , Galinhas , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/metabolismo , Proteínas do Envelope Viral/imunologia , Animais , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Herpesvirus Galináceo 1/imunologia , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/imunologia , Organismos Livres de Patógenos Específicos , Vacinas Virais/imunologia
15.
J Virol Methods ; 171(1): 264-71, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21111004

RESUMO

Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P<0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks.


Assuntos
Vírus da Doença Infecciosa da Bursa/classificação , Vírus da Doença Infecciosa da Bursa/genética , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Temperatura de Transição , Virologia/métodos , Animais , Galinhas/virologia , Primers do DNA/genética , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Dados de Sequência Molecular , Análise de Sequência de DNA
16.
Avian Dis ; 54(3): 990-3, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20945778

RESUMO

Avian nephritis virus (ANV) is thought to infect poultry flocks worldwide, but no confirmed case has been reported in Australia. The first such case is described in this study. Cases of young chickens with clinical signs of dehydration and diarrhea were submitted to our laboratory and histopathology detected interstitial nephritis. Vaccine strains of infectious bronchitis virus were detected in some of these cases but were not considered to be the causative agent. A total of seven fresh submissions from broiler chicken flocks were collected at 8-11 days of age. Degenerate PCR primers were designed based on published ANV polymerase gene sequences and used to analyze historic cases as well as the fresh submissions. Six of the seven fresh submissions, and one historic case, were positive for ANV with nucleotide sequencing confirming these results. These results establish ANV as an infectious pathogen circulating in Australian poultry.


Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/isolamento & purificação , Galinhas , Doenças das Aves Domésticas/virologia , Sequência de Aminoácidos , Animais , Infecções por Astroviridae/epidemiologia , Infecções por Astroviridae/virologia , Austrália/epidemiologia , Sequência de Bases , Rim/patologia , Rim/virologia , Dados de Sequência Molecular , Doenças das Aves Domésticas/epidemiologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
18.
J Clin Microbiol ; 47(2): 311-21, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19036935

RESUMO

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens.


Assuntos
Infecções por Adenoviridae/veterinária , Adenoviridae/classificação , Adenoviridae/isolamento & purificação , Proteínas do Capsídeo/genética , DNA Viral/genética , Doenças das Aves Domésticas/virologia , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Animais , DNA Viral/química , Genótipo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Aves Domésticas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura de Transição
19.
Avian Dis ; 50(1): 28-34, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16617977

RESUMO

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.


Assuntos
Genes Virais/genética , Herpesvirus Galináceo 1/genética , Herpesvirus Galináceo 1/isolamento & purificação , Polimorfismo de Fragmento de Restrição , DNA Viral/análise , DNA Viral/genética , Variação Genética , Herpesvirus Galináceo 1/classificação , Filogenia , Reação em Cadeia da Polimerase
20.
Avian Dis ; 46(2): 405-11, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12061651

RESUMO

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.


Assuntos
Anticorpos Antibacterianos/biossíntese , Vacinas Bacterianas/imunologia , Galinhas , Ensaio de Imunoadsorção Enzimática/veterinária , Mycoplasma/imunologia , Testes de Aglutinação/veterinária , Animais , Anticorpos Antibacterianos/isolamento & purificação , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/prevenção & controle , Infecções por Mycoplasma/veterinária , Oviposição , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/prevenção & controle , Sensibilidade e Especificidade , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia
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