Herpesvirus cyprini: detection of the viral genome by in situ hybridization

Abstract. A method of in situ hybridization with biotinylated probes is described for the spceific detection of the Herpesvirus cyprini (CHV) genome, both in CHV infected cell cultures and tissue specimens prepared from carp fry infected with the same virus. From molecularly cloned PstI-fragments of CHV DNA, two fragments were selected as probes for in situ hybridization which had no detectable homology, either with DNA of fathead minnow (FHM) cells used for virus propagation, or with the DNA of several fish herpes-viruses. The method of in situ hybridization was based on a system using streptavidin-biotinylated alkaline phosphatase for the detection of hybridization.     

An improved in situ hybridization method for the detection of fish pathogens

A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.     

Application of fluorescence in situ hybridization (FISH) techniques to fish genetics: a review

The technology is now in place for major advances in the genetics and cytogenetics of fishes at the molecular level. One promising method with broad application is fluorescence in situ hybridization (FISH). Methodologies of FISH and the current and potential uses of these chromosomal techniques in fish genetics are reviewed. Highly repetitive ribosomal genes (rDNAs) and the multicopy genes for histones have been localized in several fish species and are providing new information on the evolution of salmonid genomes. Microdissection techniques such as those used to examine the Y chromosome of lake trout can produce paint probes useful in determining chromosomal arm homologies between closely related species. Repetitive sequences isolated from various fish species have been localized to centromeres, telomeres, and sex chromosomes. Some of these are currently being used as species-specific, chromosome-specific or sex-specific probes in aquaculture of fishes. Centromeric and telomeric probes have been used to examine intraspecific chromosome rearrangements such as the transposition of rDNA. Similarly, these types of probes could be used as genome markers for examining interspecific hybrids and in the study of nuclear organization (i.e. the spatial arrangement of chromosomes in interphase cells and gametes) Chromosome mapping of single copy genes, microsatellite loci and syntenic gene groups is now possible with FISH techniques and will be useful in isolating quantitative trait loci (QTL) of importance in aquaculture.     

Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method

ABSTRACT:   Bacterial populations in goldfish feces were characterized by the fluorescent in situ hybridization (FISH) method. A total of nine different group-specific rRNA-targeted oligonucleotide probes were used. Approximately half of the microbial cells (57.8 ± 16.7%) were detected with a probe EUB338 and found to be bacteria. The microbial cells in 33–35 of the 35 samples from five specimens strongly hybridized with probes ALF1b, BET42a and GAM42a, suggesting that goldfish intestinal bacteria are mainly composed of α, β and γ-subclasses of Proteobacteria. The fact that a probe AER66 reacted with 25.6 ± 14.2% of the total microbial cells in all 35 samples, demonstrated that genus Aeromonas was the dominant species in the goldfish intestines. Genus Bacteroides including Bacteroides type A detected with a probe BAC303 was observed in 15 of 35 samples while other taxonomic groups determined with HGC69a, CF39a and P72 were detected in 6–11 of 35 samples. These results strongly suggest that Bacteroides shows the greatest daily fluctuation and interindividual variation in the intestines of goldfish. Moreover, the FISH method was proven to be useful for rapid enumeration of taxonomic groups of fish intestinal bacteria.     

鱼类神经坏死病毒实时荧光RT-PCR检测方法的建立和应用

根据GenBank中登录的鱼类神经坏死病毒CP基因序列,选择高度保守区域设计引物和TaqMan荧光探针,通过对实时荧光RT-PCR反应条件进行优化,建立了用于检测鱼类神经坏死病毒的实时荧光RT-PCR方法。利用该方法检测鱼类神经坏死病毒及其他多种常见的水生动物RNA病毒,结果只能检测到目的病毒,表明其具有良好的特异性。灵敏性试验发现,其最低检测限可达1．2pg／μL的总RNA。与RT-PCR的灵敏度对比试验表明,其敏感度比RT-PCR高100倍。对同一样品进行检测,在组内及组间的变异系数分别为0．9％以及1．5％,证实其重复性极好,并且从抽提核酸到得出结果仅需4h。对临床500份样品进行鱼类神经坏死病毒检测,结果发现有40份阳性样品。这些结果表明,本研究所建立的实时荧光RT-PCR能对鱼类神经坏死病毒进行准确、快速的检测,具有特异性好、灵敏度高的优点,是开展鱼类神经坏死病的临床检测和疫情监测工作的有力工具。[中国水产科学,2008,15（3）：506-510]     

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.     

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp,Cyprinus carpio L. using in situ hybridization

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.     

Detection of Penaeus monodon-type baculovirus (MBV) infection in Penaeus monodon Fabricius by in situ hybridization

Abstract. A digoxigenin-labelled DNA probe was used for in situ detection of the Penaeus monodon -type baculovirus (MBV) derived from cloned MBV polyhedrin genome in cultured Penaeus monodon Fabricius. First, the specificity of the probe against MBV DNA with dot blot hybridization analysis was verified. This probe indicated that cloned MBV polyhedrin fragment can be used as an MBV-specific probe. This was then used to microscopically examine sections of MBV-infected tissues for a blue-purple precipitate indicative of a positive reaction for MBV. MBV-positive cells were located only in the epithelium of the hepatopancreatic tubules and of the midgut. Furthermore, comparison of the susceptibility to MBV infection among several life-stages of the shrimp showed that the MBV genome was found in the zoea, mysis, post-larva, and adult stages, whereas MBV DNA was not detected in either eggs or nauplii. The results were quantified from in situ hybridization with an image analyser to compare the degree of cell infection among groups of cultured P. monodon collected from various farms in Taiwan.     

Quantification by fluorescent in situ hybridization of bacteria associated with Litopenaeus vannamei larvae in Mexican shrimp hatchery

The bacterial composition in the hatchery at Unidad Experimental Peñasco (UEP) of the Sonora University, Mexico, was studied by using Fluorescent In Situ Hybridization (FISH) with rRNA-specific oligonucleotide probes. We applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine shrimp culture hatchery related bacteria. Quantitative whole-cell hybridization experiments using α-, γ- and δ-Proteobacteria, and high and low G + C Gram-positive bacteria accounted for 20.8 ± 3.4% to 69.3 ± 3.3% of the total 4′,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study, marine high G + C and γ-Proteobacteria predominated in different shrimp life sub-stages. The elevated percent of high G + C and γ-Proteobacteria, extending from nauplii to mysis stages, suggest that they represent a large and significant fraction of the total picoplankton biomass in Litopenaeus vannamei larval culture.     

Serratia marcescens: a potential pathogen for fish

Abstract. Characterization of a red pigmented enterobacterium isolated from natural population of white perch, Morone americanus (Gmelin), during the course of a bacteriological survey in the Back River (Baltimore, Maryland, USA) indicated that the bacterium belonged to the species Serratia marcescents. The virulence properties of this isolate (RB 469), studied in comparison with the reference strain of S. marcescens ATCC 1800 and S. plymuthica K1R, revealed that all the strains were highly pathogenic for fish with LD₅₀s raging from 5 × 10³ to 1 × 10⁵. Similarly, te extracellular products (ECP) from the three isolates were lethal for fish (LD₅₀ ranging from 0·22 to 4·8 μg protein g^-1 fish). However, only ECP from strains with strong proteolytic activity (the white perch isolate and S. plymuthica ) were cytotoxic for both in fish and homoiothermic cell cultures and both activities were completely destroyed by heating at 100°C for 10min. In contrast, only the two S. marcescens strains which possessed phospholipase active were pathogenic for mice and produced enterotoxins. None of the Serratia strains displayed dermonecrotic factor in rabbits. All these lindings indicate that a direct relationship between eytotoxicity and virulence cannot be established.     

Sound discrimination of two sympatric,threatened fish species allows for their in situ mapping

1. Human impacts on marine ecosystems are increasing and the number of fish species listed in the Red List is constantly growing. In the Mediterranean Sea, seven of the 10 bony fishes defined as Threatened by the IUCN are known to be vocal, including the target species of this study: the shi drum (Umbrina cirrosa Linnaeus, 1758) and the brown meagre (Sciaena umbra Linnaeus, 1758). As a result, non-invasive passive acoustic monitoring (PAM) can be used to pinpoint their distribution at sea. However, for PAM to be effective, reliable acoustic discrimination is required because the sounds they emit during reproduction are remarkably similar (i.e. short broadband pulsed sounds).
2. The shi drum and the brown meagre are closely related, elusive, vocal sciaenids, partially overlapping in their ecological niche. During summer 2019, three PAM surveys were conducted along the tidal inlets of the Venice lagoon (Italy). Here, the calls of both species have been recognized according to their temporal features: shi drum sounds were made up of a lower number of longer pulses with a different envelope, repeated at a lower rate than those of the brown meagre. Further, shi drum and brown meagre sounds of different origins (aquaculture and semi-natural conditions) were analysed and compared with those collected during the survey of the study area in order to validate the field collected data.
3. Call discrimination allowed for a fine-scale species mapping, showing a partially overlapping distribution of the two species along the inlets. This is the first case in which two sciaenids have been documented to share their reproductive habitat in the Mediterranean Sea.
4. This study demonstrates that it is feasible to acoustically monitor the target species even in those parts of the Mediterranean Sea where they co-exist. This, in its turn, could provide managers with the required data for effective conservation measures to be implemented.

     

鱼类精氨酸需求研究进展

精氨酸是迄今研究的所有鱼类的必需氨基酸。近年来,在确定鱼类精氨酸需要量方面取得了巨大的进展。然而,仍然有许多养殖鱼类对精氨酸的需求有待研究。在已有的报道中,鱼类精氨酸需求存在种内和种间差异,而且这种差异较大。这篇论文综述了鱼类精氨酸需求的研究进展,包括五方面的内容,即鱼类对精氨酸的需要量,饲料中精氨酸含量对鱼类的影响,影响鱼类精氨酸需要量的因素,评价鱼类精氨酸需要量所采用的指标以及精氨酸和赖氨酸之间的相互关系。     

Markers for selection of disease resistance in fish: a review

Aquaculture is the fastest growing food producing sector in the world. However, diseases result in a major loss in production. Among the several management techniques, selective breeding for resistant trait is one of the most reliable methods and provides long-term control over disease problems. Field survival or challenge survival records provide initial information of resistant stocks. However, selection programmes demand a proper marker which can identify the resistant stock easily with much accuracy. Several immunological indicators associated with resistance to a particular disease have been reported. These include positively correlated markers such as level of natural and specific antibody, respiratory burst activity of phagocytes and level of acute-phase ceruloplasmin protein and negatively correlated markers such as serum lysozyme, haemolytic and haemagglutination activities. However, for few fish species and pathogen-specific markers viz., myeloperoxidase activity, cortisol and glucose levels, macrophage aggregation and the level of plasma proteins, a confined conclusion of association has not been established. Besides immunological markers, molecular markers are in use because of their reliable screening methods. Genome can be scanned for molecular markers which provide the full linkage map of a species or stock. Association of these molecular markers with resistant traits gives rise to quantitative trait loci (QTL) for resistance to a particular pathogen. Screening of resistant stock can be carried out using these markers. Among the several marker systems, amplified fragment length polymorphism, microsatellites and single-nucleotide polymorphism play a major role in the process of selection for disease resistance. With the use of these marker techniques, we can avoid challenge testing where a huge number of animals were being killed. Multiple trait selection can also be possible with multiple QTL identification for resistance to two or more diseases simultaneously.     

Mast cells in the gills and intestines of naturally infected fish: evidence of migration and degranulation

Immunopathological and ultrastructural studies were carried out on the gills of bream, Abramis brama, naturally infected with the copepod Ergasilus sieboldi and on the intestine of brown trout, Salmo trutta, infected with the acanthocephalan Echinorhynchus truttae. Infected gills showed extensive tissue damage due to copepod attachment, including hyperplasia, as well as proliferation of mast cells, rodlet cells and mucous cells. In parasitized gills of bream, mast cells were more abundant than in uninfected gills. They were free within the lacunae, as well as outside and inside the blood vessels of the primary lamellae, and made intimate contact with vascular endothelial cells and with neutrophils. In some infected gills, degranulation of mast cells was common. Infected intestines of brown trout had more mast cells than uninfected intestines, and these cells were often in close proximity to, and inside, the capillaries and lying close to fibroblasts of the muscularis layer and stratum granulosum. Intense degranulation of mast cells was encountered in all intestinal layers, especially near the E. truttae body.