Assuntos
Amebíase/veterinária , Amebozoários/isolamento & purificação , Doenças dos Peixes/parasitologia , Linguados , Brânquias/parasitologia , Salmo salar , Amebíase/parasitologia , Amebozoários/genética , Amebozoários/fisiologia , Animais , Brânquias/patologia , Reação em Cadeia da Polimerase , África do SulRESUMO
Laser-capture microdissection and immunohistochemistry were used to show that gene and protein expression varied in different cell types in the gills of Atlantic salmon Salmo salar, with chloride cells found to express high levels of sodium potassium ATPase and mucous cells expressing elevated levels of anterior gradient protein. It is therefore important that studies of gene expression in gill tissue take account of the proportion of the various cell types present.
Assuntos
Brânquias/citologia , Imuno-Histoquímica , Microdissecção e Captura a Laser , Salmo salar/genética , Aclimatação , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Brânquias/metabolismo , Mucoproteínas/genética , Mucoproteínas/metabolismo , Salmo salar/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Formaldehyde-based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross-linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover DNA and mRNA from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (AGD) has been reliably diagnosed from histological examination of gills although complementary methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR) are required to confirm the presence of Neoparamoeba perurans, the causative agent of AGD. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon, Salmo salar L., gills. Neutral-buffered formalin and seawater Davidson's, formaldehyde-based fixatives commonly used in fish histopathology, were compared to formalin-free commercial fixatives PAXgene®, HistoChoice™MB* and RNAlater™. Each fixative was assessed by a suite of analyses used to demonstrate AGD including routine histochemical stains, immunohistochemical stains, ISH and DNA extraction followed by PCR. All five fixatives were suitable for histological examination of Atlantic salmon gills, with seawater Davidson's providing the best quality histopathology results. Of the fixatives evaluated seawater Davidson's and PAXgene® were shown to be the most compatible with molecular biology techniques. They both provided good DNA recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and PAXgene® appear to be the best fixation methods for diagnosis and research on AGD in Atlantic salmon gills.
Assuntos
Amebíase/veterinária , Doenças dos Peixes/diagnóstico , Salmo salar/parasitologia , Fixação de Tecidos/métodos , Amebíase/diagnóstico , Amebíase/patologia , Amebozoários/fisiologia , Animais , Doenças dos Peixes/patologia , Brânquias/patologia , Fixação de Tecidos/normasRESUMO
Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Koch's postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Koch's postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature.
Assuntos
Amebíase/veterinária , Amebozoários/crescimento & desenvolvimento , Amebozoários/patogenicidade , Doenças dos Peixes/parasitologia , Brânquias/parasitologia , Salmo salar/parasitologia , Amebíase/parasitologia , Amebíase/patologia , Amebozoários/isolamento & purificação , Animais , Austrália , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Doenças dos Peixes/patologia , Brânquias/patologia , Histocitoquímica , Parasitologia/métodos , Reação em Cadeia da PolimeraseRESUMO
We report what we believe to be the first experimental demonstration of coherent beam combining of two fiber amplifiers in a 100 ns pulse regime using a signal leak between the pulses. Pulses of â¼100 W stimulated-Brillouin-scattering limited peak power are combined with 95% efficiency, a residual phase error of λ/27, and no significant beam quality degradation.