Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 70
Filtrar
1.
Dis Aquat Organ ; 99(3): 169-77, 2012 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-22832715

RESUMO

Twelve captive magnificent tree frogs Litoria splendida and 2 green tree frogs L. caerulea on a property in the Darwin rural area (Northern Territory, Australia) either died or were euthanased after becoming lethargic or developing skin lesions. Samples from both species of frog were submitted for histopathology and virus isolation. An irido-like virus was cultured from tissue samples taken from both species and was characterised using electron microscopy, restriction enzyme digests and nucleic acid amplification and sequencing. The isolates were determined to belong to the genus Ranavirus, were indistinguishable from each other and shared a 98.62% nucleotide similarity and a 97.32% deduced amino acid homology with the Bohle iridovirus over a 1161 bp region of the major capsid gene. This is the first isolation of a ranavirus from amphibians in the Northern Territory and the first report of natural infection in these 2 species of native frog. The virus is tentatively named Mahaffey Road virus (MHRV).


Assuntos
Anuros/virologia , Infecções por Vírus de DNA/veterinária , Ranavirus/classificação , Ranavirus/isolamento & purificação , Viroses/veterinária , Animais , Sequência de Bases , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/virologia , DNA Viral/genética , Dissecação , Lasers , Microscopia Eletrônica de Transmissão , Northern Territory/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Baço/patologia , Baço/ultraestrutura , Baço/virologia , Viroses/epidemiologia , Viroses/virologia
2.
J Fish Dis ; 34(2): 87-101, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21158870

RESUMO

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first isolation of KHV from koi and common carp in Indonesia and initial characterization of the isolates. Clinical signs, histopathology and virion morphology are similar to those of isolates from other countries. Phylogenetic analyses using the thymidine kinase gene amplified from each isolate and from carp tissue samples collected from KHVD outbreaks throughout Indonesia indicated that the Indonesian isolates are more closely related to the Asian than the European KHV lineage. Sequence analysis of two other variable regions between ORF29 and ORF31 (marker I) and near the start of ORF 133 (marker II) indicated that all Indonesian isolates displayed a marker I allele (I(++)) previously identified only in isolates of the Asian lineage. However, in the marker II region, all Indonesian isolates displayed the II(-) allele, which has been reported previously only amongst isolates of the European lineage, and nine of these displayed a mixed genotype (II(+)II(-)). The I(++)II(-) genotype has not been reported previously and appears to represent a new intermediate lineage that may have emerged in Indonesia.


Assuntos
Carpas/virologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/veterinária , Herpesviridae/genética , Herpesviridae/isolamento & purificação , Timidina Quinase/genética , Animais , Aquicultura , Carpas/fisiologia , Linhagem Celular , DNA Viral/análise , DNA Viral/classificação , DNA Viral/genética , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/fisiopatologia , Proteínas de Peixes/classificação , Proteínas de Peixes/genética , Herpesviridae/classificação , Herpesviridae/patogenicidade , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/fisiopatologia , Infecções por Herpesviridae/virologia , Indonésia/epidemiologia , Dados de Sequência Molecular , Filogenia , Timidina Quinase/classificação , Vírion/ultraestrutura
3.
Science ; 287(5452): 443-9, 2000 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-10642539

RESUMO

Emerging infectious diseases (EIDs) of free-living wild animals can be classified into three major groups on the basis of key epizootiological criteria: (i) EIDs associated with "spill-over" from domestic animals to wildlife populations living in proximity; (ii) EIDs related directly to human intervention, via host or parasite translocations; and (iii) EIDs with no overt human or domestic animal involvement. These phenomena have two major biological implications: first, many wildlife species are reservoirs of pathogens that threaten domestic animal and human health; second, wildlife EIDs pose a substantial threat to the conservation of global biodiversity.


Assuntos
Animais Selvagens , Doenças Transmissíveis/veterinária , Ecossistema , Agricultura , Animais , Animais Domésticos , Clima , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/etiologia , Doenças Transmissíveis/transmissão , Conservação dos Recursos Naturais , Reservatórios de Doenças , Humanos , Zoonoses
4.
Dis Aquat Organ ; 73(3): 175-92, 2007 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-17330737

RESUMO

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world's amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis in the future.


Assuntos
Anuros/microbiologia , Quitridiomicetos/isolamento & purificação , Micoses/veterinária , Reação em Cadeia da Polimerase/veterinária , Animais , Quitridiomicetos/genética , DNA Fúngico/análise , Etanol/farmacologia , Técnicas Imunoenzimáticas/veterinária , Larva/microbiologia , Micoses/diagnóstico , Micoses/patologia , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/isolamento & purificação , Temperatura , Dedos do Pé/microbiologia , Microbiologia da Água
5.
Microbes Infect ; 3(4): 297-306, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11334747

RESUMO

The ultrastructure of Hendra and Nipah viruses is described in cultured cells, pigs, horses and humans. Differences in ultrastructure between the viruses are evident within infected cell cultures and lungs from infected amplifier hosts. These differences are important in viral identification and differentiation and understanding the pathogenesis of disease.


Assuntos
Infecções por Paramyxoviridae/virologia , Paramyxovirinae/ultraestrutura , Animais , Encéfalo/virologia , Células Cultivadas , Doenças dos Cavalos/virologia , Cavalos , Humanos , Pulmão/virologia , Microscopia Eletrônica , Infecções por Paramyxoviridae/veterinária , Suínos , Doenças dos Suínos/virologia
6.
J Immunol Methods ; 178(1): 1-12, 1995 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-7530266

RESUMO

We describe the use of a gene-targeted random epitope library for the mapping of antigenic determinants. A DNA clone encoding the target antigen was digested randomly with DNase I to generate a population of DNA fragments of different sizes and sequences. After size fractionation, small DNA fragments (100-200 bp) were isolated and cloned into the phage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of the phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epitope libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Using this approach, we have mapped a monoclonal antibody (mAb)-defined epitope on the bluetongue virus outer capsid protein VP5. This epitope is not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although the example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy described should be equally applicable to genes of unknown sequence and for screening of epitopes using polyclonal antibodies. The approach can also be extended to identify immunodominant epitope from much more complex genome-targeted random epitope library for virus, bacteria and eukaryotic organisms. Other applications of recombinant phages expressing defined immunodominant epitopes include serodiagnosis and vaccine development.


Assuntos
Antígenos Virais/imunologia , Vírus Bluetongue/imunologia , Capsídeo/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais/genética , Antígenos Virais/ultraestrutura , Bacteriófagos/genética , Sequência de Bases , Vírus Bluetongue/genética , Vírus Bluetongue/ultraestrutura , Capsídeo/genética , Capsídeo/ultraestrutura , Proteínas do Capsídeo , Epitopos/genética , Vetores Genéticos/genética , Coloide de Ouro , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Seleção Genética , Análise de Sequência de DNA
7.
Virus Res ; 43(1): 1-15, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8822630

RESUMO

The ultrastructure of the equine morbillivirus (EMV) which was implicated in the death of one human and fourteen horses in Queensland, Australia during September 1994 and a 36 year old man from Queensland in October 1995 is described. The ultrastructure of the virus and the intracellular virus-specific structures are characteristic for the family Paramyxoviridae. Cytoplasmic nucleocapsids were observed within the infected cells monolayers, endothelial cells (lung) of infected horses and the neurons within the brain of the 36 year old Queensland man. Aggregates of smaller nucleocapsid-like structures were also observed within the brain of the same man; these did not react with sera from recovered EMV-infected horses or from a recovered EMV-infected human. Co-examination of rinderpest virus (RPV), bovine parainfluenza-3 (BPIV-3), human respiratory virus (HRSV) and Sendai virus revealed that their envelope-associated surface projections are equivalent in length to the 15 nm spikes of EMV. EMV differed from these other viruses in that the majority of virions possessed surface projections of two distinct lengths (18 and 15 nm). Further ultrastructural examinations of plaque purified EMV revealed a small percentage of EM viruses possessed a mixed array of surface projections indicating that the 'double-fringed' (DF) particles may be the result of a post-translational modification(s).


Assuntos
Infecções por Morbillivirus/virologia , Morbillivirus/ultraestrutura , Adulto , Animais , Encéfalo/patologia , Encéfalo/virologia , Bovinos , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Equidae/virologia , Cavalos/virologia , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Infecções por Morbillivirus/patologia , Infecções por Morbillivirus/veterinária , Nucleocapsídeo/ultraestrutura , Paramyxoviridae/ultraestrutura , Células Vero
8.
Virus Res ; 32(3): 313-28, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8079513

RESUMO

The host protective antigen gene VP2 of infectious bursal disease virus (IBDV) was genetically modified and expressed by recombinant fowlpox viruses (rFPV). To achieve cell surface localization, VP2 was expressed as a hybrid protein with signal sequence and membrane anchors of influenza virus hemagglutinin or neuraminidase. Native VP2 was expressed as VP2 alone or as self-processing VP2-VP4-VP3 polyprotein for coexpression of IBDV structural proteins. VP2 hybrid protein containing the carboxy-terminal membrane anchor sequence of influenza virus hemagglutinin was located on the cell surface and was N-glycosylated. The expression of VP2 fused to the N-terminal signal/anchor sequence of influenza virus neuraminidase led to cell lysis and the VP2 protein remained mainly unglycosylated. Cell surface localization of VP2 reduced immunogenicity (antibody induction) and abolished protection in poultry in comparison with the native VP2 expressed by FPV as VP2 alone or as the self-processing VP2-VP4-VP3. Vaccination of poultry with rFPV expressing native VP2 protein alone provided better protection from IBDV infection than VP2 derived from the VP2-VP4-VP3 polyprotein.


Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Varíola das Aves Domésticas/imunologia , Vírus da Doença Infecciosa da Bursa/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Infecções por Birnaviridae/prevenção & controle , Linhagem Celular , Membrana Celular/imunologia , Galinhas , Vírus da Varíola das Aves Domésticas/genética , Glicoproteínas/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Doenças das Aves Domésticas/prevenção & controle , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Organismos Livres de Patógenos Específicos , Vacinação , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vacinas Virais/genética
9.
Virus Res ; 54(2): 165-87, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9696125

RESUMO

A novel lyssavirus isolated from Pteropid bats in Australia (Australian Bat Lyssavirus, ABLV) has been characterised using gene sequence analyses, electron microscopy and a panel of monoclonal antibodies. Electron microscopic examination of Pteropid bat and mouse brain material as well as virus isolated from tissue culture medium, showed the presence of bullet-shaped rhabdovirus particles and structures characteristic of lyssavirus. Analysis using nucleocapsid (N) specific monoclonal antibodies, showed a strong relationship between this new lyssavirus and serotype 1 rabies. The nucleotide sequence of the prototype strain of ABLV was determined from the initiator methionine codon for the nucleocapsid protein (N protein) to the amino terminus of the polymerase gene (L protein), a distance of 5344 nucleotides. Comparisons of the deduced N, phosphoprotein (P), matrix protein (M), and glycoprotein (G) proteins showed that ABLV was more closely related to serotype 1 classic rabies viruses than to other members of the Lyssavirus genus. The percent relatedness of the ABLV proteins when compared to the cognate proteins of PV (Pasteur vaccine strain) rabies was 92, 75, 87 and 75% for the N, P, M and G proteins, respectively. Phylogenetic studies of N protein sequences showed clearly that ABLV is an unrecognised member of the Lyssavirus genus and represents a new genotype, genotype 7.


Assuntos
Quirópteros/virologia , Lyssavirus/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos Virais/imunologia , Austrália , Sequência de Bases , Linhagem Celular , Cricetinae , DNA Viral , Glicoproteínas/genética , Lyssavirus/genética , Lyssavirus/imunologia , Lyssavirus/ultraestrutura , Camundongos , Dados de Sequência Molecular , Nucleocapsídeo/genética , Fosfoproteínas/genética , Filogenia , Vírus da Raiva/imunologia , Análise de Sequência de DNA , Proteínas da Matriz Viral/genética
10.
Science ; 288(5475): 2319b-20b, 2000 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-17769839
11.
J Virol Methods ; 19(1): 23-32, 1988 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2450888

RESUMO

Competitive studies, involving neutralizing monoclonal antibodies (NTmAbs) to outercoat proteins of Akabane virus were performed by immunoelectron microscopy. The experiments were designed to determine whether the NTmAbs were directed against the same or spatially different epitopes. Characteristics of NTmAbs in direct and indirect gold-labelling studies were determined. It was found that the protein A method gave cross-contamination of the immuno-gold complexes whereas direct conjugation of the NTmAbs to gold probes gave clean, specific and intense labelling. Analysis of dilution curves confirmed that saturation of antigenic sites did not occur and secondly determined the optimum working dilutions for the conjugated probes. The data generated in the preliminary studies enabled reliable results to be obtained from the double-labelling competitive experiment. We found that the 2 NTmAbs were directed to either the same epitope or to 2 separate but neighbouring epitopes where the binding of one NTmAb inhibited the binding of the second. The results demonstrate that if reliable data is to be obtained in double-labelling immunoelectron microscopical studies then experiments must be meticulously designed.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Microscopia Eletrônica/métodos , Ligação Competitiva , Bunyaviridae/imunologia , Epitopos/imunologia , Ouro , Técnicas Microbiológicas , Proteínas do Envelope Viral/imunologia
12.
J Virol Methods ; 46(2): 117-32, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8188809

RESUMO

The immuno-gold-silver staining (IGSS) technique was used in scanning electron microscopy for the detection and semi-quantitation of low copy antigens on the surface of cells. The methodology was exploited in experiments designed to examine the interaction of small numbers of virus particles with the surface of susceptible host cells. Using bluetongue virus (BTV) as an example, IGSS procedures confirmed that maximum adsorption occurred within 60 min and that adsorbed virus particles were distributed randomly on the surface of the cell. Neutralising antibody did not prevent binding of BTV to the plasma membrane, but abrogated virus uptake. The use of IGSS in the study of virus-cell interactions was validated by transmission electron microscopy and classical biochemical experiments utilising radioactively-labelled virus.


Assuntos
Antígenos Virais/análise , Vírus Bluetongue/imunologia , Adsorção/efeitos dos fármacos , Animais , Anticorpos Antivirais/farmacologia , Vírus Bluetongue/ultraestrutura , Células Cultivadas , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia Eletrônica de Varredura
13.
Acta Trop ; 78(2): 103-16, 2001 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-11230820

RESUMO

By using the criteria that define emerging infectious diseases (EIDs) of humans, we can identify a similar group of EIDs in wildlife. In the current review we highlight an important series of wildlife EIDs: amphibian chytridiomycosis; diseases of marine invertebrates and vertebrates and two recently-emerged viral zoonoses, Nipah virus disease and West Nile virus disease. These exemplify the varied etiology, pathogenesis, zoonotic potential and ecological impact of wildlife EIDs. Strikingly similar underlying factors drive disease emergence in both human and wildlife populations. These are predominantly ecological and almost entirely the product of human environmental change. The implications of wildlife EIDs are twofold: emerging wildlife diseases cause direct and indirect loss of biodiversity and add to the threat of zoonotic disease emergence. Since human environmental changes are largely responsible for their emergence, the threats wildlife EIDs pose to biodiversity and human health represent yet another consequence of anthropogenic influence on ecosystems. We identify key areas where existing expertise in ecology, conservation biology, wildlife biology, veterinary medicine and the impact of environmental change would augment programs to investigate emerging diseases of humans, and we comment on the need for greater medical and microbiological input into the study of wildlife diseases.


Assuntos
Doenças dos Animais/etiologia , Animais Selvagens/microbiologia , Doenças Transmissíveis Emergentes/etiologia , Doenças dos Animais/epidemiologia , Doenças dos Animais/microbiologia , Animais , Animais Selvagens/parasitologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/microbiologia , Ecologia , Meio Ambiente , Humanos , Zoonoses/epidemiologia , Zoonoses/etiologia , Zoonoses/microbiologia
14.
Vet Microbiol ; 58(2-4): 135-43, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9453125

RESUMO

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.


Assuntos
Búfalos/virologia , Bovinos/virologia , Vírus da Doença Hemorrágica Epizoótica/classificação , Infecções por Reoviridae/veterinária , Ovinos/virologia , Animais , Linhagem Celular , Vírus da Doença Hemorrágica Epizoótica/isolamento & purificação , Vírus da Doença Hemorrágica Epizoótica/ultraestrutura , Japão , Microscopia Eletrônica , Northern Territory , Filogenia , Infecções por Reoviridae/fisiopatologia , Infecções por Reoviridae/virologia , Sorotipagem
15.
Comp Immunol Microbiol Infect Dis ; 17(3-4): 163-88, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8001343

RESUMO

The general properties of the orbiviruses have been examined at the physical, structural and molecular level. At the structural level, the orbiviruses (with the exception of the Kemerovo serogroup) appear similar. The replicative events are also similar, however differences in the ultrastructure of virus-specific structures and their association with components of the host cell have been observed. Further research in this area may be used to differentiate between the serogroups and even some serotypes, of orbiviruses. At the molecular level the properties of the genome can be used to determine relationships between members of the orbivirus genus. These relationships are revealed using a variety of techniques including serology and gene sequence analysis. Not only are the different serological responses to gene products present in the mature virus particle used for differential diagnosis, but the gene sequences themselves can also be utilized. Understanding of the relationships between these viruses is progressing to the point that insights into orbivirus molecular epidemiology is now possible.


Assuntos
Orbivirus/fisiologia , Orbivirus/ultraestrutura , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Orbivirus/genética , Filogenia , Replicação Viral/fisiologia
16.
Micron ; 25(6): 597-605, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7881897

RESUMO

Cells infected with bluetongue virus (BTV) were prepared for immunocytochemistry by freeze substitution, the progressive lowering of temperature technique and the Tokuyasu method. Sections containing virus-infected cells were incubated with specific monoclonal antibodies and colloidal gold probes to detect virus antigens of varying copy number; these BTV proteins were structural proteins VP2 and VP7 and the non-structural protein NS2. Protocols compared in this study represented those used in laboratories which handle infectious agents and as such, all samples were pre-fixed with minimum concentrations of glutaraldehyde to inactivate the virus. No statistical difference was found between the gold-labelling of sections prepared by the progressive lowering of temperature technique and freeze substitution. The results showed that cryo-sections yielded the best signal-to-noise ratio for all proteins examined in this study and were therefore the most sensitive system for the detection of low copy number proteins. The data and associated inferences relate to the system described in this paper and possibly other analogous systems.


Assuntos
Antígenos Virais/análise , Vírus Bluetongue/imunologia , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão e Varredura/métodos , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Capsídeo/análise , Proteínas do Capsídeo , Linhagem Celular , Criopreservação , Crioultramicrotomia , Substituição ao Congelamento , Corpos de Inclusão Viral/ultraestrutura , Suínos , Proteínas do Core Viral/análise , Proteínas não Estruturais Virais/análise
17.
Dis Aquat Organ ; 39(2): 151-4, 2000 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-10715821

RESUMO

The extraction and amplification of nucleic acid from formalin-fixed and paraffin-embedded tissues has become an important exercise in the collection of retrospective epidemiological data. A protocol is described that enables the extraction and amplification of dsDNA from fixed tissues within paraffin blocks and from specimens stored in 10% (aq) formalin. The procedure can be used for the examination of ranavirus DNA within archival tissues thereby providing valuable data for identifying the origin and tracing the spread of ranaviruses.


Assuntos
DNA Viral/isolamento & purificação , Ranavirus/genética , Animais , Boidae/virologia , Fixadores , Formaldeído , Parafina , Percas/virologia , Reação em Cadeia da Polimerase/veterinária
18.
Dis Aquat Organ ; 60(2): 141-8, 2004 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-15460858

RESUMO

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade.


Assuntos
Anuros/microbiologia , Quitridiomicetos/genética , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Primers do DNA , DNA Ribossômico/genética , Técnicas Histológicas , Dados de Sequência Molecular , Sensibilidade e Especificidade , Análise de Sequência de DNA
19.
Dis Aquat Organ ; 43(1): 1-14, 2000 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-11129376

RESUMO

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.


Assuntos
Infecções por Birnaviridae/veterinária , Birnaviridae/isolamento & purificação , Doenças dos Peixes/virologia , Oncorhynchus mykiss , Salmo salar , Animais , Anticorpos Monoclonais , Aquicultura , Birnaviridae/genética , Birnaviridae/patogenicidade , Birnaviridae/ultraestrutura , Infecções por Birnaviridae/patologia , Infecções por Birnaviridae/virologia , Western Blotting/veterinária , Efeito Citopatogênico Viral , Primers do DNA/química , DNA Viral/química , DNA Viral/isolamento & purificação , Doenças dos Peixes/patologia , Histocitoquímica , Técnicas Imunoenzimáticas/veterinária , Vírus da Necrose Pancreática Infecciosa/patogenicidade , Microscopia Eletrônica/veterinária , Testes de Neutralização/veterinária , Pâncreas/patologia , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência , Análise de Sequência de DNA , Tasmânia
20.
Dis Aquat Organ ; 56(1): 59-64, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-14524502

RESUMO

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide. It has been associated with catastrophic declines of frog populations including those in pristine habitats in Queensland, Australia. To facilitate genetic and disease studies of this fungus and related species, it is essential to have a reliable long-term storage method to maintain genetic integrity of isolates. We have adapted well-established techniques used for the long-term storage of tissue-culture cell lines to the preservation of B. dendrobatidis and other chytridiomycetes. This simple method has allowed us to recover these fungi from storage at -80 degrees C and in liquid nitrogen over an extended period. With this technique it is now possible to preserve saprobic and parasitic isolates from a variety of environmental and disease situations for comparative genetic and biological studies.


Assuntos
Quitridiomicetos , Criopreservação/métodos , Quitridiomicetos/ultraestrutura , Crioprotetores/química , Microscopia Eletrônica , Nitrogênio/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA