RESUMO
Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Gado , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/veterinária , Proteína Estafilocócica A/genética , SuínosRESUMO
The sheep body louse, Bovicola ovis (B. ovis), is one of the most significant ectoparasites affecting Australia's sheep flocks. Despite this, detection methods for B. ovis infestation are limited to visual inspection and ELISA. A colourimetric loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of B. ovis DNA. Diagnostic sensitivity and specificity of LAMP were compared with those of visual inspection and PCR and validated using field samples collected from 22 farms. Two different DNA extraction methods using a commercial kit and a boiling method were also compared. The highest sensitivity and specificity were observed when PCR was used and DNA was extracted using a commercial kit. Compared with PCR, the LAMP assay demonstrated a sensitivity and specificity of 90% and 92% when DNA was extracted by a commercial kit and 100% and 75% when DNA was extracted by the boiling method, respectively. The LAMP test developed in this study could potentially serve as a point-of-care diagnostic tool for monitoring of sheep flocks as well as surveillance of B. ovis populations.
Assuntos
Testes Diagnósticos de Rotina/normas , Infestações por Piolhos/veterinária , Técnicas de Amplificação de Ácido Nucleico/normas , Doenças dos Ovinos/diagnóstico , Animais , Austrália , Colorimetria , Iscnóceros/genética , Infestações por Piolhos/diagnóstico , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/parasitologiaRESUMO
The establishment of viral pathogens in new host environments following spillover events probably requires adaptive changes within both the new host and pathogen. After many generations, signals for ancient cross-species transmission may become lost and a strictly host-adapted phylogeny may mimic true co-divergence while the virus may retain an inherent ability to jump host species. The mechanistic basis for such processes remains poorly understood. To study the dynamics of virus-host co-divergence and the arbitrary chances of spillover in various reservoir hosts with equal ecological opportunity, we examined structural constraints of capsid protein in extant populations of Beak and feather disease virus (BFDV) during known spillover events. By assessing reservoir-based genotype stratification, we identified co-divergence defying signatures in the evolution BFDV which highlighted primordial processes of cryptic host adaptation and competing forces of host co-divergence and cross-species transmission. We demonstrate that, despite extensive surface plasticity gathered over a longer span of evolution, structural constraints of the capsid protein allow opportunistic host switching in host-adapted populations. This study provides new insights into how small populations of endangered psittacine species may face multidirectional forces of infection from reservoirs with apparently co-diverging genotypes.
Assuntos
Doenças das Aves/genética , Infecções por Circoviridae/genética , Circovirus/genética , Evolução Molecular , Animais , Doenças das Aves/virologia , Proteínas do Capsídeo/genética , Infecções por Circoviridae/virologia , Circovirus/patogenicidade , Fluxo Gênico , Genótipo , Especificidade de Hospedeiro/genética , Papagaios/genética , Papagaios/virologia , Filogenia , Psittaciformes/genética , Psittaciformes/virologiaRESUMO
Like other avian circovirus species, Pigeon circovirus (PiCV) is known to be genetically diverse with a relatively small circular single-stranded DNA genome of 2â kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Recent paleoviral evidence hints towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and dispersal of their hosts. Limited availability of PiCV genome sequence data in Australia has hindered phylogeographic studies in this species, so we screened clinically normal rock doves (Columba livia) in regional New South Wales, and demonstrated a high prevalence (76%) of PiCV infection by PCR. We also recovered 12 complete novel PiCV genomes and phylogenetic analyses revealed that PiCV circulating in Australian feral pigeons formed two strongly supported monophyletic clades. One clade resided with PiCV genomes from Poland, Australia, United Kingdom, Belgium, China, and Japan, and another basal clade was more closely related to PiCV genomes from Poland. A novel more distantly-related PiCV rep gene formed a solitary clade with weak posterior support. So we further analysed all selected partial rep gene sequences to demonstrate a likely naturally occurring spillover infection from a passerine circovirus candidate. The findings suggest that there is a high degree of genetic variation within PiCV in Columbiformes with potential greater admixture between avian circoviruses within Australia than previously known. RESEARCH HIGHLIGHTS Confirmed high prevalence rate of PiCV circulating in Australian wild pigeons. Highlighted extensive recombination events within Australian PiCV. Demonstrated a likely naturally occurring spillover infection from a passerine circovirus candidate.
Assuntos
Doenças das Aves/epidemiologia , Infecções por Circoviridae/veterinária , Circovirus/genética , Columbidae/virologia , Genoma Viral/genética , Recombinação Genética , Animais , Doenças das Aves/virologia , Proteínas do Capsídeo/genética , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , New South Wales/epidemiologia , Filogenia , Reação em Cadeia da Polimerase/veterináriaRESUMO
The presence of endogenous viral elements in host genomes hints towards much older host-virus relationships than predicted by exogenous phylogenies, with highly mutable single-stranded DNA (ssDNA) viruses and RNA viruses often occupying entangled multispecies ecological niches. The difficulty lies in unravelling the long-term evolutionary history of vertebrate virus-host relationships and determining the age of a potentially ancient tree based only fresh shoots at the tips. Resolving such lineages, and the sometimes great discrepancy amongst evolutionary timescales, is problematic, especially when purifying selection or recombination can significantly alter the accuracy of phylogenetic reconstruction methods. Pathogens which occupy entangled multispecies ecological niches add a further layer of complexity but we show that multi-host scenarios may also provide opportunities to identify allopatric or sympatric paleobiological signals that can unlock longer term phylogenies. We identified host-based, cryptic, sympatric differentiation in beak and feather disease virus in the Psittaciformes tribe Loriini along with endogenous circovirus motifs in Kea (Nestor notabilis) and Gondwanan vicariance estimates to infer the evolutionary timescale of the circoviruses. This demonstrated a chronology of psittacine circovirus speciation aligned to conservative Zealandic divergences for relic circovirus motifs in Kea and a 10million year divergence coinciding with the Papuan central range orogeny that triggered the radiation of Loriini and segregation of an antecedent viral clade in Australian lorikeets. Estimates of circovirus speciation in birds highlighted a Gondwanan dominant group in Neoaves with passerine, columbid and larid circoviruses deeply separated from those in waterfowl, consistent with a Triassic divergence of Galloanserae. The circovirus tree had a deep ancestry in invertebrates with a Palaeozoic expansion in fish and mammals. We show that longer term evolutionary relationships in viruses which have a high rate of mutation and admixture can be disentangled, highlighting that contemporary virus host-switching can be explained by deep intra-lineage host phylogeny.
Assuntos
Doenças das Aves/virologia , Infecções por Circoviridae/veterinária , Circovirus/genética , Papagaios/genética , Animais , Doenças das Aves/genética , Infecções por Circoviridae/virologia , Evolução Molecular , Genes Virais , Variação Genética , Genoma Viral , Dados de Sequência Molecular , Papagaios/virologia , Filogenia , Seleção Genética , Tropismo ViralRESUMO
Mycoplasma synoviae infections result in significant economic losses in the chicken and turkey industries. A commercially available live temperature-sensitive (ts (+)) vaccine strain MS-H has been found to be effective in controlling M. synoviae infections in commercial layer and broiler breeder farms in various countries, including Australia. Detection and differentiation of MS-H from field strains (ts (-)) and from ts (-) MS-H reisolates in vaccinated flocks is vital in routine flock status monitoring. At present microtitration is the only available technique to determine the ts phenotype of M. synoviae. This technique is time consuming and not amenable to automation. In the present study, a quantitative real-time polymerase chain reaction (Q-PCR) was combined with simultaneous culturing of M. synoviae at two different temperatures (33°C and 39.5°C) to determine the ts phenotype of 22 Australian M. synoviae strains/isolates. The M. synoviae type strain WVU-1853 was also included for comparison. A ratio of the copy numbers of the variable lipoprotein haemagglutinin (vlhA) gene at the two temperatures was calculated and a cut-off value was determined and used to delineate the ts phenotype. In all M. synoviae strains/isolates tested in this study, the ts phenotype determined using Q-PCR was in agreement with that determined using conventional microtitration. Combination of Q-PCR with differential growth at two different temperatures is a rapid, reliable and accurate technique that could be used as an effective tool in laboratories actively involved in ts phenotyping of M. synoviae strains/isolates.
Assuntos
Monitoramento Epidemiológico/veterinária , Infecções por Mycoplasma/veterinária , Mycoplasma synoviae/crescimento & desenvolvimento , Fenótipo , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/microbiologia , Temperatura , Animais , Austrália , Primers do DNA/genética , Regulação da Expressão Gênica/genética , Infecções por Mycoplasma/epidemiologia , Mycoplasma synoviae/metabolismo , Aves Domésticas , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
The microbial communities that inhabit the intestinal tract play an important role in modulating health and productivity. Environmental stressors can impact microbial communities, which can significantly influence host physiology. Cattle are subjected to several environmental stressors when placed on feedlots, such as transportation stress, exposure to feedlot environments and change in diet and management. Exposure to these stressors could influence host gut microbiota, which in turn, could potentially influence host health and performance. The aim of the current study was to characterise the temporal changes that occur in intestinal microbiota as a consequence of feedlot placement by profiling 16s rRNA sequences in rectal faecal samples. When faecal microbiome profiles were compared in terms of relative abundances and alpha diversity metrics, the results showed significant, observable changes in profiles 2 days post-feedlot induction. Furthermore, beta-diversity analysis indicated that the phylogenetic similarity between samples significantly decreased on day 2 (PERMANOVA, p < 0.001). These trends were suggestive of a short-term reduction in microbial diversity coupled with decreased similarity between animals. These changes warrant further investigation and could provide opportunities for improved performance, health and even welfare of feedlot cattle in future.
RESUMO
Human milk oligosaccharides (HMOs) are the third most abundant solid component after lactose and lipids of breast milk. All mammal milk contains soluble oligosaccharides, including neutral milk oligosaccharides (NMOs) without sialic acid (Sia) moieties and acidic oligosaccharides or sialylated milk oligosaccharides (SMOs) with Sia residues at the end of sugar chains. The structural, biological diversity, and concentration of milk oligosaccharides in mammalian milk are significantly different among species. HMOs have multiple health benefits for newborns, including development of immune system, modification of the intestinal microbiota, anti-adhesive effect against pathogens, and brain development. Most infant formulas lack oligosaccharides which resemble HMOs. Formula-fed infants perform poorly across physical and psychological wellbeing measures and suffer health disadvantages compared to breast-fed infants due to the differences in the nutritional composition of breast milk and infant formula. Of these milk oligosaccharides, SMOs are coming to the forefront of research due to the beneficial nature of Sia. This review aims to critically discuss the current state of knowledge of the biology and role of SMOs in human milk, infant formula milks, and milk from several other species on gut and brain health of human and animal offspring.
RESUMO
Spotty liver disease (SLD) is a bacterial disease of chicken, causing mortalities and reduction in egg production, hence, contributing to economic loss in the poultry industry. The causative agent of SLD has only recently been identified as a novel Campylobacter species, Campylobacter hepaticus. Specific primers were designed from the hsp60 gene of Campylobacter hepaticus and PCR followed by high-resolution melt curve analysis was optimised to detect and differentiate three species of Campylobacter (Campylobacter coli, Campylobacter jejuni and Campylobacter hepaticus). The three Campylobacter species produced a distinct curve profile and was differentiated using HRM curve analysis. The potential of the PCR-HRM curve analysis was shown in the genotyping of 37 Campylobacter isolates from clinical specimens from poultry farms. PCR-HRM curve analysis of DNA extracts from bile samples or cultures from bile samples, were identified as Campylobacter hepaticus and confirmed by DNA sequencing. The DNA sequence analysis of selected samples from each of the three HRM distinctive curves patterns showed that each DNA sequence was associated with a unique melt profile. The potential of the PCR-HRM curve analysis in genotyping of Campylobacter species was also evaluated using faecal specimens from 100 wild birds. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Campylobacter species using either bacterial cultures or clinical specimens.
Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter/genética , Campylobacter/isolamento & purificação , Animais , Doenças das Aves/etiologia , Doenças das Aves/microbiologia , Campylobacter/patogenicidade , Infecções por Campylobacter/microbiologia , Galinhas/genética , DNA/química , Primers do DNA/genética , Fígado/microbiologia , Hepatopatias/veterinária , Desnaturação de Ácido Nucleico/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Aves Domésticas/genética , Doenças das Aves Domésticas/microbiologia , Análise de Sequência de DNA/métodosRESUMO
Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines. In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vlhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG strains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respiratory swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Mycoplasma gallisepticum/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Proteínas de Bactérias/genética , Sequência de Bases , Primers do DNA/genética , Dados de Sequência Molecular , Mycoplasma gallisepticum/classificação , Mycoplasma gallisepticum/genética , Temperatura de TransiçãoRESUMO
Conservation efforts for the orange-bellied parrot (Neophema chrysogaster), one of the world's most critically endangered bird species, have been hampered by beak and feather disease virus (BFDV) spillover infection. To understand the vulnerability of orange-bellied parrots to potential reservoirs of infection we investigated geographic versus taxonomic structure in 160 full-genome and 319 partial Rep gene BFDV sequences from captive and wild orange-bellied parrots and other wild parrot species in Australia. We found that Australian BFDV populations are structured by host taxonomy. By identifying genetic stratification of BFDV in reservoir hosts we characterized three separate recent incursions of BFDV into orange-bellied parrots from other wild parrots, which demonstrates the susceptibility of critically endangered species to multiple threats of pathogen re-emergence. Our study highlighted how loss of endemic circulating BFDV in orange-bellied parrots precipitated repeated spillover into an immunologically naïve population, causing significant disease.
Assuntos
Doenças das Aves/virologia , Infecções por Circoviridae/veterinária , Circovirus , Espécies em Perigo de Extinção , Papagaios/virologia , Animais , Austrália/epidemiologia , Doenças das Aves/epidemiologia , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Conservação dos Recursos Naturais , Doenças Endêmicas , Genoma Viral , Genótipo , FilogeniaRESUMO
This paper describes the genomic architecture of a complete mitogenome from a red-bellied parrot (Poicephalus rufiventris). The mitogenome sequence was circular and 15,524 bp in length. Compared to other Psittacidae species, the genome encoded a conserved structure consisting of 13 protein-coding genes (PCGs), two rRNA genes, 21 tRNA genes, and two control regions, however, the mitogenome was missing a part of control region and a tRNA-Leu. The lengths of 12S and 16S ribosomal RNA were 975 bp and 1569 bp, respectively. The overall base composition of the mitogenome of P. rufiventris was dominated by higher AT (51.6%) than GC (48.4%) content. The complete mitogenome sequence determined in this study would be useful to track the deeper evolutionary history and the conservation of P. rufiventris.
RESUMO
The complete genome sequence of beak and feather disease virus (BFDV) from a purple crowned lorikeet (Glossopsitta porphyrocephala) was characterized. The genome consists of 2,010 nucleotides and encodes replicase-associated protein and capsid protein. This is the first evidence of BFDV infectivity and complete genome sequence for this novel host.
RESUMO
Three complete genomes of beak and feather disease virus (BFDV) were recovered from wild musk lorikeets (Glossopsitta concinna). The genomes consisted of 2,008 to 2,010 nucleotides (nt) and encode two major proteins transcribing in opposing directions. This is the first report of BFDV complete genome sequences obtained from this host species.
RESUMO
The complete genome sequence of beak and feather disease virus (BFDV) from a fledgling red-capped parrot (Purpureicephalus spurius) was assembled and characterized. The genome consists of 1,995 nucleotides and encodes two major proteins in opposing directions. This is the first evidence of BFDV infectivity and a complete genome sequence for this novel host.
RESUMO
Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10(-4) ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population.
Assuntos
Coinfecção/veterinária , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/genética , Doenças das Aves Domésticas/diagnóstico , Perus/microbiologia , Adesinas Bacterianas/genética , Animais , Coinfecção/diagnóstico , Coinfecção/microbiologia , Genes Bacterianos , Lectinas/genética , Técnicas de Diagnóstico Molecular , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Reação em Cadeia da Polimerase , Doenças das Aves Domésticas/microbiologia , Temperatura de TransiçãoRESUMO
Since the characterization of psittacine beak and feather disease (PBFD) in 1984, a wide range of avian circoviruses have been discovered with varying pathogenic effects amongst a diverse range of avian hosts. Until recently these circovirus species were thought to be restricted to within avian Orders such as the Psittaciformes for beak and feather disease virus (BFDV) and Columbiformes for pigeon circovirus with little evidence of cross-family transmission or replication. We report evidence of a naturally occurring novel host switch event with self-limiting BFDV infection in a group of rainbow bee-eaters (Merops ornatus) a species of Coraciiformes unrelated to parrots and not previously known to be susceptible to any avian circovirus. The outbreak highlights important and unexpected aspects of disease emergence and host-switching pertinent to other situations when viruses might cross species boundaries as well as the potential of avian circoviruses to infect disparate host species.
RESUMO
Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.
Assuntos
Campylobacter coli/genética , Campylobacter jejuni/genética , Código de Barras de DNA Taxonômico/métodos , DNA Bacteriano/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas de Bactérias/genética , Infecções por Campylobacter/microbiologia , Campylobacter coli/classificação , Campylobacter jejuni/classificação , DNA Bacteriano/química , Variação Genética , Genótipo , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Especificidade da Espécie , Temperatura de TransiçãoRESUMO
Structural insights into the biology of viruses such as beak and feather disease virus (BFDV) which do not replicate in cell cultures are increasingly reliant on recombinant methods for protein production and purification. Development of efficient methods for homogenous production of BFDV capsid protein is also essential for vaccine development and diagnostic purposes. In this study, two different plasmids (pMCSG21 and pMCSG24), three homologous BFDV capsid proteins, and two unique expression media (auto-induction and IPTG-induced expression) were trialled for over-expression of the BFDV in Escherichia coli. Over-expression was observed for all three recombinant targets of BFDV capsid protein using E. coli BL21 (DE3) Rosetta 2 cell lines under IPTG induction. These proteins could be purified using an optimized, two-step purification process using a buffer containing 20mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 500 mM NaCl and supplemented with 200 mM L-arginine at pH 10.5, to yield a soluble and stable protein of greater than 95% purity. The final concentration of purified protein was approximately fourteen-to-eighteen fold greater than that reported previously. Initial crystallization and X-ray diffraction confirm that the protein is structured in a manner consistent with icosahedral symmetry. Antigenicity of recombinant Cap was confirmed by immunoassay, verifying its validity for use in continued experimentation as a potential DNA vaccine, a reagent in diagnostic assays, and purified concentrated protein for structural and functional biology.
Assuntos
Proteínas do Capsídeo/isolamento & purificação , Circovirus/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Meios de Cultura/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Plasmídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR-HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories.