RESUMEN
In Australia, disease caused by betanodavirus has been reported in an increasing number of cultured finfish since the first report of mortalities in 1990. Partial coat protein gene sequences from the T2 or T4 regions of 8 betanodaviruses from barramundi Lates calcarifer, sleepy cod Oxyeleotris lineolata, striped trumpeter Latris lineata, barramundi cod Cromileptes altivelis, Australian bass Macquaria novemaculata and gold-spotted rockcod Epinephelus coioides from several Australian states were determined. Analysis of the 606 bp nucleotide sequences of the T2 region of 4 isolates demonstrated the close relationship with isolates from the red-spotted grouper nervous necrosis virus (RGNNV) genotype and the Cluster Ia subtype. Comparison of a smaller 289 bp sequence from the T4 region identified 2 distinct groupings of the Australian isolates within the RGNNV genotype. Isolates from barramundi from the Northern Territory, barramundi, sleepy cod, barramundi cod and gold-spotted rockcod from Queensland, and striped trumpeter from Tasmania shared a 96.2 to 99.7% nucleotide identity with each other. These isolates were most similar to the RGNNV genotype Cluster Ia. Isolates from Australian bass from New South Wales and from barramundi from South Australia shared a 98.6% sequence identity with each other. However, these isolates only shared an 85.8 to 87.9% identity with the other Australian isolates and representative RGNNV isolates. The closest nucleotide identity to sequences reported in the literature for the New South Wales and South Australian isolates was to an Australian barramundi isolate (Ba94Aus) from 1994. These 2 Australian isolates formed a new subtype within the RGNNV genotype, which is designated as Cluster Ic.
Asunto(s)
Enfermedades de los Peces/virología , Nodaviridae/genética , Nodaviridae/aislamiento & purificación , Filogenia , Infecciones por Virus ARN/veterinaria , Animales , Australia/epidemiología , Enfermedades de los Peces/epidemiología , Peces , Infecciones por Virus ARN/epidemiología , Infecciones por Virus ARN/virologíaRESUMEN
AIMS: Vibrio harveyi is an important pathogen, causing potential devastation to marine aquaculture. This organism, however, is extremely difficult to identify because it is phenotypically diverse. Biochemical identification can involve many tests and take weeks to perform. The aim of this work is to develop a PCR that can reduce the number of biochemical tests, and the time taken, to get a definitive identification of this organism. METHODS AND RESULTS: The PCR was developed using 16S rDNA sequences from a number of V. harveyi strains, and other vibrios. The described test gave positive results for all strains of V. harveyi tested. However, some strains of V. alginolyticus also gave positive results and a small number of biochemical tests were required to differentiate between these two species. This indicated that preisolation of the bacteria was needed and therefore the test was not applicable to the testing of mixed populations directly. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of identification of this species was significantly reduced from a number of weeks to a few days. Hence, diagnosis of affected animals will be faster and earlier treatment can be administered which may increase the survival rate from vibriosis.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Vibriosis/diagnóstico , Vibrio/clasificación , Microbiología del Agua , Animales , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Vibrio/genética , Vibrio/aislamiento & purificación , Vibriosis/veterinariaRESUMEN
AIMS: To determine the complete nucleotide sequence of the bacteriophage VHML and establish a hypothesis for the virulence conversion caused by VHML infection of Vibrio harveyi. METHODS AND RESULTS: The complete nucleotide sequence of VHML was determined (43,193 bp) and used to identify putative genes. The translated products of these genes were compared with reported sequences to assign hypothetical functions. All anticipated structural genes and putative genes for lysogeny were identified. In addition, we found a complete N6-adenine methyltransferase (Dam) gene that appeared to have an essential site for ADP-ribosylating toxins at the C-terminal of the translated product. CONCLUSIONS: Virulence conversion of V. harveyi by VHML may be associated with Dam transcriptional regulation. The Dam gene may also encode for a toxin component similar to ADP-ribosylating toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript lays the foundation for understanding the virulence of toxin-producing V. harveyi. Further research into aspects discussed here will lead to a greater comprehension regarding the invertebrate disease vibriosis and its control in the farming of these animals.
Asunto(s)
Genoma Viral , Transformación Bacteriana , Vibrio/virología , Toxinas Bacterianas/biosíntesis , Secuencia de Bases , Datos de Secuencia Molecular , Vibrio/metabolismo , Vibrio/patogenicidad , Vibriosis/microbiología , Virulencia/genéticaRESUMEN
Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans. Despite their importance, their modes of virulence have yet to be fully elucidated. Here, we present a previously unreported bacteriophage extracted from a toxin-producing strain of V. harveyi isolated from moribund prawn larvae in tropical Australia. Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double-stranded linear DNA). We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like). VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V. harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested). The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed.
Asunto(s)
Toxinas Bacterianas , Bacteriófagos/clasificación , Vibrio/virología , Animales , Australia , Toxinas Bacterianas/biosíntesis , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/ultraestructura , ADN Viral/análisis , Vibrio/metabolismoRESUMEN
Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.
Asunto(s)
Aeromonas hydrophila/genética , Sondas de ADN/genética , Aeromonas hydrophila/clasificación , Southern Blotting , ADN Bacteriano/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Técnica del ADN Polimorfo Amplificado Aleatorio , Especificidad de la EspecieRESUMEN
Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) uses arbitrary primers and low stringency annealing conditions to amplify anonymous DNA fragments which are then depicted in agarose gels. RAPD-PCR fingerprints have been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals. The analysis of such fingerprints is based upon the assumption that co-migration of amplicons does not occur and that any given band contains a single amplicon. This report shows that co-migration of fragments of nearly identical size, but different nucleotide sequences, occurs between different isolates and within single RAPD-PCR bands from Aeromonas hydrophila. The possibility of the same phenomenon occurring for other prokaryotic or eukaryotic genomes argues for caution in the interpretation of RAPD-PCR fingerprints.
Asunto(s)
Aeromonas hydrophila/genética , Dermatoglifia del ADN , ADN Bacteriano/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Aeromonas hydrophila/clasificación , Secuencia de Bases , Southern Blotting , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida. DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous. However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer. salmonicida. Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences. All five fragments were evaluated as DNA probes for the specific detection of Aer. salmonicida DNA: two hybridized specifically to DNA of all Aer. salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida, achromogenes and masoucida, but not subspecies smithia; one hybridized to subspecies salmonicida and achromogenes, but not subspecies masoucida or smithia; and one hybridized to subspecies salmonicida, achromogenes and smithia, but not subspecies masoucida. It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens.
Asunto(s)
Aeromonas/genética , Sondas de ADN/genética , Animales , Southern Blotting/métodos , Enfermedades de los Peces/diagnóstico , Peces/microbiología , Reacción en Cadena de la Polimerasa/métodos , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
A data file consisting of 40 biochemical and physiological tests for the differentiation of aeromonads was constructed based upon existing published data from various geographical locations and laboratories. The data file covers all the current genomospecies (or hybridisation groups) of Aeromonas and is therefore applicable to bacteriologists from a wide variety of fields. The protocol derived from the data file was adapted for use with a semi-interactive computer identification program. This program was challenged with the type strains of all the currently recognised genomospecies and each genomospecies was correctly identified, with only one strain (ATCC 7966, HG1) having an identification probability of less than 90%. The program was also tested with environmental and clinical isolates and again identifications were achieved with high probabilities. This protocol is designed for use on presumptive isolates of Aeromonas spp. and can be carried out in routine laboratories not equipped to perform the complex DNA hybridisation techniques which are currently used for the differentiation of genomospecies of Aeromonas.
Asunto(s)
Aeromonas/clasificación , Aeromonas/metabolismo , Infecciones por Bacterias Gramnegativas/microbiología , Aeromonas/aislamiento & purificación , Animales , Enfermedades de los Peces/microbiología , Peces/microbiología , Infecciones por Bacterias Gramnegativas/veterinaria , HumanosRESUMEN
Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to investigate the differentiation of the genus Aeromonas at the genomospecies level. Of 20 primers evaluated, six produced profiles which contained multiple bands capable of differentiating the genomospecies. These six primers were also used in RAPD-PCR analyses of clinical and environmental isolates of the different genomospecies. In most cases, each strain gave a unique fingerprint, illustrating genetic heterogeneity at the genomospecies level. However, some homogeneity in fragment sizes was seen among strains within a genomospecies which was not apparent in strains from different genomospecies. This study therefore complements and supports the current classification of Aeromonas into genomospecies. These results also show that RAPD-PCR has the potential to differentiate between the genomospecies of Aeromonas.
Asunto(s)
Aeromonas/clasificación , Genoma Bacteriano , Técnica del ADN Polimorfo Amplificado Aleatorio , Aeromonas/genética , Animales , Secuencia de Bases , Dermatoglifia del ADN , Cartilla de ADN , ADN Bacteriano/genética , ADN Bacteriano/normas , Peces/microbiología , Heterogeneidad Genética , Datos de Secuencia Molecular , Estándares de Referencia , Microbiología del AguaRESUMEN
Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei, both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris. Commonly expressed glycosidase activities were alpha-D-galactosidase and beta-N-acetyl-D-glucosaminidase followed by beta-D-glucosidase and alpha-L-fucosidase. The combined production of beta-N-acetyl-D-glucosaminidase and alpha-D-galactosidase was a feature of the endocarditis isolates. In contrast, beta-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris. The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aminopeptidasas/metabolismo , Endocarditis Bacteriana/etiología , Glicósido Hidrolasas/metabolismo , Lactobacillus/enzimología , Coagulación Sanguínea , Humanos , Lactobacillus/patogenicidad , Especificidad de la EspecieRESUMEN
Lactobacilli are often considered to be commensal or beneficial participants in human microbial ecology and considerable research is being carried out into the effects of the use of lactobacilli as additives in both human and animal diets. However, lactobacilli also cause some human diseases (e.g. dental caries, rheumatic vascular disease, septicaemia and infective endocarditis (IE)), and have recently been identified as potential emerging pathogens in elderly and immunocompromised patients, particularly those receiving broad spectrum antibiotic therapy. The identification of potential pathogenic traits amongst lactobacilli will therefore facilitate the use of the organisms for probiotic purposes. The ability to aggregate human platelets is considered to be a possible pathogenic trait in the progression of IE. A comparison of bacterial cell surface properties amongst L. rhamnosus strains showed that platelets were aggregated by 5/5 IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains the respective numbers were 2/5 and 2/9. However two strains, morphological mutants of a non-aggregating strain, which had been re-isolated after passaging through rats were found to aggregate platelets. No loss of aggregating function occurred on extensive subculturing of IE strains. Aggregation also occurred with 11/14 strains for five other species, namely, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salvivarius, with each species being represented indicating that the property is not uncommon in the genus. A comparison of IE and oral isolates of L. rhamnosus and L. paracasei subsp. paracasei and seven other Lactobacillus species, has shown that the binding of both fibronectin and fibrinogen by lactobacilli is greatly increased, up to 50 fold, when the pH is reduced from 7.0 to 5.0. Re-exposing the lactobacilli to a neutral pH environment releases most of the bound proteins, but the amount still remaining bound to the cell is several times more than is bound at neutral pH. Lactobacilli will also bind to the proteins that make up the extracellular matrix of endothelial cells. Lactobacilli bound significantly better to collagen types I and V than to types III and IV (p < 0.01). Further, strains isolated from IE cases, particularly L. rhamnosus strains, bound significantly better to types I and V than did 'normal' strains (p < 0.02). Type V collagen has been demonstrated at the sites of endothelial damage. Thus the binding of lactobacilli, particularly L. rhamnosus to these collagen types may be of importance in the early stages of colonization of the damaged heart valve.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Microbiología de Alimentos , Lactobacillus/patogenicidad , Animales , Adhesión Bacteriana , Colágeno/metabolismo , Endocarditis Bacteriana/etiología , Endocarditis Bacteriana/microbiología , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Infecciones por Bacterias Grampositivas/etiología , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Técnicas In Vitro , Lactobacillus/aislamiento & purificación , Lactobacillus/fisiología , Agregación Plaquetaria , Especificidad de la EspecieRESUMEN
Aggregation of platelets by bacteria is a potential factor in the pathogenesis of infective endocarditis. Twenty-five strains from the Streptococcus sanguis group, including 15 recent isolates from cases of endocarditis, were compared for their ability to aggregate human and rat platelets over periods of 15 and 25 min, respectively. In each case, 76% of strains caused aggregation; the median time to onset of aggregation was longer for human platelets (12 min) than for rat platelets (1 min). Strains unable to aggregate human platelets included three from cases of endocarditis. There was no correlation between the ability to aggregate human and rat platelets, although the majority of strains (60%) aggregated both. Tests on representative strains for their ability to aggregate rabbit platelets gave results similar to those for rat platelets, including a median time of 1 min to onset of aggregation. The differences in the ability of individual bacterial strains to aggregate human and animal platelets indicate that caution is needed in extrapolating in-vitro observations to the in-vivo situation.
Asunto(s)
Endocarditis Bacteriana/microbiología , Boca/microbiología , Agregación Plaquetaria , Infecciones Estreptocócicas/microbiología , Streptococcus sanguis/fisiología , Animales , Modelos Animales de Enfermedad , Humanos , Conejos , RatasRESUMEN
Lancefield group C Streptococcus milleri group (SMG) strains are the only SMG types that are able to aggregate human platelets. Complete aggregation occurred within 10 min of mixing bacterial cells and platelets together in the ratio 8:1. Substances which (i) chelated cations; (ii) inhibited the cycloxygenase pathway in platelets; (iii) reduced the availability of ADP and disrupted platelet membrane stability; (iv) reduced bacterial aggregation of platelets. The platelet-interacting substance on the surface of the SMG appeared to be proteinacious as digestion of bacterial cells with protease inhibited aggregation whereas treatment with lipase, periodate or antisera to Lancefield group C polysaccharide had no effect.
Asunto(s)
Agregación Plaquetaria/fisiología , Streptococcus/patogenicidad , Absceso/etiología , Endocarditis Bacteriana/etiología , Humanos , Técnicas In Vitro , Especificidad de la Especie , Infecciones Estreptocócicas/etiología , Streptococcus/clasificación , Streptococcus/fisiologíaRESUMEN
The ability to aggregate human platelets was examined for five Lactobacillus rhamnosus strains and five Lactobacillus paracasei subsp. paracasei strains isolated from patients with infective endocarditis (IE), 25 laboratory isolates from the same two species, and 14 strains from five other oral species, namely Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius. Amongst the L. rhamnosus strains, platelets were aggregated by all five IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains, the respective numbers were 2/5 and 2/9. Aggregation also occurred with 11/14 strains of the other five species; each species was represented. The optimal ratio of bacteria to platelets for aggregation was approximately 1:1, and there was considerable variation in the lag phase that preceded aggregation, depending on the source of the platelets. Overall, the lag phase varied between 0.25 +/- 0.1 and 20.4 +/- 3.2 min and the percentage aggregation ranged between 70 +/- 2.6 and 104 +/- 13.5%. Confirmation that aggregation was being observed came from studies with five strains on the inhibitory effects of EDTA, dipyridamole, apyrase, imipramine, acetylsalicylic acid and quinacrine. Inhibition of aggregation by L. rhamnosus strains by the peptide arginine-glycine-aspartic acid-serine (RGDS) further indicated a role for fibronectin and/or fibrinogen. Pronase treatment of cells for 1 h and extraction of bacterial surface components with 0.1 M-Tris/HCl (pH 8.5) at 37 degrees C for 1 h stopped aggregation in 8/9 IE strains. Extracted surface proteins (200 micrograms) completely inhibited platelet aggregation by 8/9 of the homologous strains.(ABSTRACT TRUNCATED AT 250 WORDS)