Automatic counting of FISH-labeled microbes by an LED illuminated detecting apparatus

ABSTRACT:   The Bioplorer (BP) (Matsushita Ecology Systems Co. Ltd, Kasugai, Aichi-ken, Japan) is an apparatus that consists of a light-emitting diode and optical and image analysis systems. This instrument has been developed and used for the semi-automatic counting of total microbes, mainly in food, cosmetics and industrial products. In this study, the applicability of BP to the detection and enumeration of eukaryotic and prokaryotic cells, after the fluorescent in situ hybridization (FISH) procedure, was examined. The cells of a yeast ( Candida albicans ) and a bacterium ( Escherichia coli ) were specifically labeled using an oligonucleotide probe with fluorochrome and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The numbers of cells after FISH treatment and DAPI staining agreed well, indicating that the BP optical device and image analysis system had enough sensitivity to detect and quantify eukaryotic and prokaryotic cells. Because of its simplicity and reliability, BP can be a new tool for quantification of specifically labeled target microorganisms, both in industrial products and natural samples.     

Rapid enumeration of bacteria grown on surimi-based products by flow cytometry

ABSTRACT: A rapid method to enumerate bacteria adhered on a surimi-based product (kamaboko) by flow cytometry (FCM) is described. To remove Escherichia coli cells from the surface of kamaboko, ultrasonic energy was used. Almost all cells can be removed from kamaboko in 3 min with ultrasonic treatment. Because the sample might contain various non-bacterial particles such as food additives and debris of products, propidium iodide was used to discriminate bacterial cells from non-bacterial particles. Fluorescence scattergrams could distinguish bacteria from the particles, and the FCM method could be used to enumerate bacteria adhered on the surface of kamaboko during storage. Cell numbers determined by FCM paralleled well with those measured using a traditional colony counting method in the range of 10⁴–10⁸ cells/g. The FCM assay could enumerate cells within 1 min and the total assay time, including sample preparation, was less than 30 min.     

Effects of replacement of fish meal with fermented cottonseed meal on growth performance,body composition and haemolymph indexes of Pacific white shrimp,Litopenaeus vannamei Boone, 1931

A feeding trial was performed for 28 days to evaluate the effects of replacement of fish meal (FM) with fermented cottonseed meal (FCM) on growth, body composition and haemolymph indexes of juvenile Litopenaeus vannamei. Four isonitrogenous and isoenergetic diets were formulated by using FCM (96.4, 206, 317 and 417 g kg^?1) to substitute 25%, 50%, 75% and 100% of FM in a control diet respectively. Each diet was randomly allotted to four tanks with 20 shrimp per tank. The feeding trail was conducted in an indoor flow‐through aquaculture system. Shrimp fed diets containing 317 and 417 g kg^?1 of FCM obtained lower (P < 0.05) final weight, weight gain, specific growth ratio, protein efficiency ratio as well as a higher (P < 0.05) feed conversion ratio compared with shrimp fed the control diet. The body ash content decreased (P < 0.05) in shrimp fed the diet with complete replacement of FM than those in other treatments. Moreover, increasing the dietary inclusion of FCM linearly raised (P < 0.05) the concentrations of total gossypol, (?) and (+) gossypol enantiomers in the whole shrimp body. No difference (P > 0.05) was observed in haematological parameters among the treatments. The results suggest that up to 50% of FM can be replaced by FCM without adverse effects on growth and feed utilization of L. vannamei.     

Snorkelling as a method for assessing spawning stock of Atlantic salmon, Salmo salar

Abstract  Reliability of underwater snorkel counts of adult Atlantic salmon, Salmo salar L., was analysed in the tributaries of the River Teno, close to the spawning period. In small (width 5–20 m) rivers, the replicated total counts of salmon were reasonably precise (CV = 5.4–8.5%), while in the medium-sized river (width 20–40 m) the precision of the counting method was considerably lower (CV = 15.3%). Low precision in a medium sized river was also observed in an experiment using marked live fish where the observation efficiency varied between 36.4% and 70.0%. In a small river, the detection efficiency of artificial fish silhouettes (test salmon) was almost perfect in pools (98%), but decreased in rapids (84%). Separate counts of males, females, grilse and large salmon were usually more variable than total counts, indicating that divers were more capable of locating a fish than properly identifying its sex and sea-age. The behaviour of adult salmon was favourable to conduct snorkel counts, as fish normally stayed still, or after hesitating, moved upstream (>95%of the cases) when encountering a diver. The high observation efficiency (>90%) and precision, favourable behaviour of salmon and congruence between snorkel counts and catch statistics in small rivers suggest that reliable data on Atlantic salmon spawning stock can be collected by snorkeling provided that the environmental conditions are suitable and the divers are experienced.     

Effects of the probiotic Bacillus subtilis (BP11 and BS11) on the growth and survival of Pacific white shrimp,Litopenaeus vannamei

The effect of Bacillus S11 (BS11)‐ and/or Bacillus P11 (BP11)‐supplemented feeds on the growth performance, survival, immunoenhancement and disease resistance of cultured Pacific white shrimp, Litopenaeus vannamei, was evaluated. Four feeding treatments of (i) regular feed (control), (ii) BS11‐supplemented feed, (iii) BP11‐supplemented feed and (iv) BS11‐and BP11‐supplemented feed were prepared and used for shrimp cultivation in closed recirculating cement tanks (~400 L) in two trials, one for juvenile and PL‐30 shrimp at 60 and 90 days, respectively. The results showed that BS11 gave a higher probiotic potential than BP11 for both age groups of L. vannamei in cultivation, because the average weight and survival of shrimp fed BS11‐supplemented feed were significantly higher (P < 0.05) than those of the control and the other two groups. The survival of shrimp fed either BS11‐or both BS11‐and BP11‐supplemented feed was significantly higher (P < 0.05) than that of the control group. In addition, the highest total haemocyte and granular haemocyte counts and phenoloxidase activity were found in shrimp fed with the BS11‐supplemented feed. After challenge with Vibrio harveyi 639 (~10⁷ CFU mL^?1) by immersion, the lowest cumulative death (%) and disease resistance were clearly found in shrimp fed with the BS11‐supplemented feed.     

The effectiveness of commercial probiotics in northern white shrimp <Emphasis Type="Italic">Penaeus vannamei</Emphasis> ponds

ABSTRACT:   The effectiveness on water quality, population density of bacteria, and shrimp production in ponds treated with commercial probiotics was tested in Penaeus vannamei ponds in Hai-yan, China. Six ponds with replicates for treatment and control were used. Results showed that the probiotics could improve the population density of beneficial bacterial flora, reduce concentrations of nitrogen and phosphorus, and increase yields of shrimp. The average counts of Bacillus sp., ammonifying bacteria, and protein mineralizing bacteria were found to be significantly higher in treated ponds compared to control ponds ( P  < 0.05). In control ponds, an increase in presumptive vibrios was observed and the average density was up to 2.09 × 10³ cfu/mL, whereas that was only 4.37 × 10² cfu/mL in treated ponds ( P  < 0.05). The use of probiotics also significantly increased dissolved oxygen ( P  < 0.05) and reduced dissolved reactive-phosphorus, total inorganic nitrogen and chemical oxygen demand ( P  < 0.05). An average of 8215 ± 265 kg shrimp/ha was obtained in treated ponds with a feed conversion ratio (FCR) of 1.13 ± 0.05 and survival rate of 81.00 ± 6.25% compared with 4985 ± 503 kg shrimp/ha, 1.35 ± 0.12 and 48.67 ± 3.51%, respectively, in control ponds. This indicates that the addition of the commercial probiotics had a noticeable influence on water quality of shrimp ponds and shrimp production.     

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g^?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.     

Use of Ox‐Aquaculture© for disinfection of live prey and meagre larvae,Argyrosomus regius (Asso, 1801)

Live prey used for marine larval fish (rotifers and Artemia) as well as intensive larval rearing conditions are susceptible to the proliferation of bacteria that are the cause for reduced growth and larval mortality. Hydrogen peroxide has been recently proved a good disinfectant in aquaculture, either for eggs, larvae or live prey. In this study the effects of a hydrogen peroxide‐based product, Ox‐Aquaculture^©, on live prey (rotifers and Artemia) and meagre larvae bacterial load, composition and final status have been tested. A 34.6% reduction of total heterotrophic bacteria and 59.7% of Vibrionaceae were obtained when rotifers were exposed for 15 min to 40 mg L^?1 of the product. A 34.3% reduction of total heterotrophic bacteria and 37.7% of Vibrionaceae were obtained when Artemia were exposed for 5 min to 8000 mg L^?1 of the product. More than 95% reduction of total heterotrophic bacteria and 75% of Vibrionaceae were obtained when meagre larvae were exposed for 1 h to 20 mg L^?1 of the product. Furthermore, disinfection of enriched live prey with the product did not change the fatty acid composition and survival of the live prey and improved final larval survival.     

Extracellular protease production of bacteriolytic bacteria isolated from marine environments

ABSTRACT:   Fourteen bacterial strains isolated from marine environments exhibited antagonistic action against a wide range of bacteria including Vibrio spp. A double layer agar method was used for preliminary screening to determine the relative degree of growth inhibition or bacteriolysis exhibited by the isolates. Most of the antagonistic isolates were found to be Gram-negative, motile rods and were oxidase positive, and oxidative in the oxidation and fermentation test, suggesting that they are belong to the genera Pseudomonas . The antagonistic isolates lyzed the dead cells of marine Gram-negative bacteria in both plate and liquid methods. Bacteriolytic and casein hydrolytic activities were observed in the culture supernatant of the isolates. Anion exchange column chromatography (Toyopearl DEAE-650 M) was used to purify the extracellular protease produced by an antagonistic strain A1-J25a. The active fractions of protease collected from the eluted solution also exhibited bacteriolytic activity.     

Efficiency of presence-absence sampling used to estimate the mean abundance of fish eggs

ABSTRACT:   Statistical properties of estimators relating to the mean abundance of fish eggs were investigated using the data from the presence-absence sampling (PAS) and counting sampling (CS). PAS, which focuses on the presence-absence of eggs in a sample, is more cost-effective yet is unlikely to give more precise estimates than CS, which counts the number of eggs. But when limitations are given on the sampling cost and number of sampling stations, PAS may have advantages. This study shows that the mean square error (MSE) of the maximum likelihood estimator (MLE) based on PAS may become smaller than the MSE of the MLE based on counting data when the number of observations for PAS becomes larger. The observation number for PAS is determined, which minimizes the MSE of a combined estimator from the two MLE under a restriction of the total cost of observation. A dual problem is also solved. It is shown that MSE of the MLE in PAS is a monotone increasing function of the oversight probability. PAS becomes more informative as the distribution of the number of eggs is more aggregated.     

Sensory and Microbial Quality of Retail Finfish With Emphasis on Antibiotic-Resistant Bacteria

The sensory and microbial quality of commercially important retail finfish of Kolkata, India was evaluated emphasizing oxytetracycline and chloramphenicol resistant bacterial contamination. The finfish had total viable counts (TVCs) in the range of log 4.30/g in Labeo bata to log 7.93/g in Mystus tengara. The counts of oxytetracycline and chloramphenicol resistant bacteria were in the range of log 2.00–6.79/g and log 1.70–5.80/g fish, respectively. About 72% of the Kolkata market finfish harbored viable bacteria exceeding the acceptable international limit of 5.0?×?10⁵/g. Up to 3.45 and 29.86% of TVCs were resistant to chloramphenicol and oxytetracycline, respectively. The marine fish sold in the Kolkata market were of marginal to inferior quality with the highest mean quality index point (QIP) range of 6.80–10.20. Most freshwater fish were of fresh to marginal quality and recorded QIP in the range of 3.80–8.20. Though the freshwater fish were judged fresh by the quality index method in comparison to the marine counterparts, they recorded high viable counts. The results of the present study call for the need to educate the fish handlers on appropriate hygiene and safe handling of fish and the public about fish-borne bacterial diseases and the impact of antibiotic resistant bacteria on human health. They also show the potential for fish to be a source of antibiotic bacteria for human consumers.     

Enhancing productivity of Labeo victorianus ponds using maize flour as a carbohydrate source

This study investigated the effects of manipulating carbon–nitrogen (C/N) ratio and fish stocking density on pond productivity: total heterotrophic bacteria counts, plankton biovolume and benthic macro‐invertebrates. Labeo victorianus juveniles were reared for 72 days in 18 hapas suspended in six ponds measuring 150 m² at densities of 10, 15 and 25 fish m^?2. Fish in hapas received a locally formulated and prepared feed containing 295 g kg^?1 crude protein, and ponds were treated with a C/N ratio of either 10 or 20. All treatments were carried out in triplicate. Increasing C/N ratio from 10 to 20 increased phytoplankton by 13% and zooplankton biovolume by 25% in the water column (P < 0.001). Total benthic macro‐invertebrates biovolumes were also 30% higher (P < 0.05) with a C/N ratio of 20 compared to 10. Total heterotrophic bacteria counts increased both in water and sediment by 29% while net yield increased by 15% from 1534 (C/N 10) to 1821 (C/N 20) kg ha^?1 72 day^?1. C/N ratio of 20 and a stocking density of 25 fish m^?2 led to the highest yield, survival, production and net benefits. It is suggested that polyculture may lead to better utilization of pond communities to further improve pond productivity.     

我国水产品全产业链损耗研究

本文通过调研水产品在流通、加工、贮藏和零售四个产业链环节的损耗情况,对不同类型水产品损耗原因和差异进行分析,提出降低我国水产品产业链损耗的建议措施.单一水产品品种的损耗率数据通过调研直接获得,水产品(整体)及各品类产业链损耗率基于2008-2019年水产品品类及品种产量结构加权计算所得.结果显示:2008-2019年,...     

Comparative activity and stability under salinity conditions of different antimicrobial peptides isolated from aquatic animals

This study reports the in vitro activity of six antimicrobial peptides (AMPs) produced by aquatic animals (most marine invertebrates): tachyplesin (Tach), magainin (Mag), clavanin (Clav), penaeidin (Pen), mytilin (Myt) and antilipopolysaccharide factor (ALF) against marine vibrios, filamentous fungi and yeast. Their stability under salinity conditions and seawater was also examined. The results showed that Mag, Myt and especially Tach and ALF (minimum inhibitory concentration<1.5 μM) had a potent activity against all tested vibrio species, whereas Clav and Pen were ineffective (up to 50 μM). With respect to the antifungal activity, each AMP had a different potency according to the fungal species. In general terms, Tach was the most potent peptide, followed by Mag. Interestingly, Tach, Myt and ALF had a significant effect on the filamentous fungus Fusarium solani that could be pathogenic to marine organisms. All AMPs had a tendency to decrease or lose their activity at high salinity (>225 mM NaCl). Tach and Myt were the most stable peptides, maintaining significant activity under seawater salinity (450 mM). Curiously, all peptides lost their effect under seawater conditions. The results suggest that Tach, ALF and Myt are the most promising candidates for potential therapeutic use in farmed-marine species, because all have a significant and broad antimicrobial activity maintained at high salinity.     

Assessing feasibility of replacement of fishmeal with enzyme‐treated feather meal in the diet of juvenile turbot (Scophthalmus maximus L.)

A feeding trial was conducted to examine the potential of enzyme‐treated feather meal (EFM) as a fishmeal substitute in the diet of turbot. Fishmeal (650 g/kg) was incrementally replaced (0, 160, 320, 480, 640 and 800 g/kg) by EFM (0, 80, 160, 240, 320, 400 g/kg) to prepare six experimental diets (Control, EFM8, EFM16, EFM24, EFM32 and EFM40). Diet EFM24 was supplemented with lysine and methionine to form seventh diet (EFM24 + AA). The eighth diet was formulated with 240 g/kg steam‐processed feather meal to replace 480 g/kg fishmeal (SFM24). Eight diets were each fed to triplicate groups of juvenile turbot (37.47 g) in a recirculating aquaculture system for 7 weeks. The results showed that fish fed diet EFM24 grew better than those fed SFM24 (p < .05). The growth performance, body composition and serum biochemical indices of fish fed diet EFM8 were not statistically different compared to the control treatment (p > .05). However, further increase in fishmeal replacement (32%–80%) affected negatively growth, nutrients digestibility, serum biochemical indices and body composition (p < .05). Supplementation of lysine and methionine (EFM24 + AA) significantly improved growth performance and diet utilization compared to EFM24 (p < .05). Altogether results suggested that the EFM could comprise 80 g/kg in a diet for juvenile turbot by concurrent reduction of the fishmeal content by 160 g/kg without lysine and methionine supplementation.     

Bacterial degradation of antibiotic residues in marine fish farm sediments of Uranouchi Bay and phylogenetic analysis of antibiotic-degrading bacteria using 16S rDNA sequences

ABSTRACT:   Antibiotic residues in marine sediments of fish farms negatively influence microbial ecologic systems. The microbial degradation of antibiotic residues was experimentally examined in the marine sediments of Uranouchi Bay, to which one of five antibiotics was added. After incubation reducing physical factors, ampicillin, doxycycline, oxytetracycline, and thiamphenicol were significantly degraded, while josamycin maintained most of the initial amounts. The isolates resistant to ampicillin, josamycin, oxytetracycline, or thiamphenicol degraded each antibiotic in wide ranges of degrees, whereas the isolates degrading doxycycline were not obtained. Microbial degradation may contribute to the disappearance of ampicillin, doxycycline, oxytetracycline, and thiamphenicol in the fish farm. In contrast, the disappearance of josamycin would depend on physical factors, but the bacteria degrading josamycin at least exist in the marine sediments. Phylogenetic analysis using 16S rDNA sequences demonstrated that the antibiotic-resistant isolates formed several clusters in the Gram-positive bacterial group, the Flavobacterium–Cytophaga–Bacteroides group, and the proteobacteria subdivisions. The antibiotic-resistant bacterial population would be composed of various species including ubiquitous coastal bacterial groups. Several species of antibiotic resistant bacteria show antibiotic degradation activities, and appear to contribute to the disappearance of antibiotics in Uranouchi Bay.     

Vitrification of sperm from marine fish: effect on motility and membrane integrity

Our goal was to develop a standardized approach for sperm vitrification of marine fish that can be applied generally in aquatic species. The objectives were to: (i) estimate acute toxicity of cryoprotectants over a range of concentrations; (ii) evaluate the properties of vitrification solutions (VS); (iii) evaluate different thawing solutions and (iv) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10‐μL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post‐thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X‐1000^? + 1% Z‐1000^? had an average post‐thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to higher osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.     

Comparison of four antibiotics with indigenous marine Bacillus spp. in controlling pathogenic bacteria from shrimp and Artemia

Use of antibiotics for the control of bacterial diseases in shrimp culture has caused several adverse impacts to the industry. This has resulted in the search for alternative environment friendly approaches to overcome bacterial infections. This study was conducted to investigate the use of beneficial bacteria as an alternative to antibiotics. Ten pathogenic bacterial species isolated from shrimp, Penaeus monodon, and Artemia cysts were tested for susceptibility to indigenous marine Bacillus subtilis AB65, Bacillus pumilus AB58, Bacillus licheniformis AB69 and compared with oxytetracycline, chloramphenicol, gentamicin and bacitracin, which are common antibiotics used in Asian aquaculture. The Bacillus spp. were isolated from the local marine environment for bioremediation use in shrimp hatcheries and were proven to reduce total ammonium nitrogen. The pathogenic bacterial isolates were 90% susceptible to B. subtilis AB65, 70% susceptible to B. pumilus AB58 and B. licheniformis AB69 and 100% susceptible to oxytetracycline, chloramphenicol and gentamicin but only 40% to bacitracin. Two representative isolates of the vibrio group, Vibrio alginolyticus VaM11 and Vibrio parahaemolyticus VpM1, when tested for competitive exclusion by a common broth method using the marine Bacillus spp., showed decreased viable counts from 10(8) to 10(2) cfu mL(-1). The results suggest that the action of the marine bacteria appears to be significant in protecting the host shrimp against pathogenic bacteria. In addition to the alternative use of antibiotics, the selected marine bacteria had additional bioremediation properties of reducing ammonia.     

A quick method for the assessment of activity and inhibition of fish amylases

A sensitive and quick method was developed to determine the presence of α-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch–agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L^–1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2–20 U well^–1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.     

海水蛭弧菌分离纯化方法初步研究

蛭弧菌(Bdellovibrio)广泛存在于自然水体,它具有噬菌的特性,对水体中细菌数量控制具调节作用,有可能是进行生物防治的一种重要生物。本文比较不同分离方法、上层培养基营养盐浓度和富集培养与直接培养等对海水蛭弧菌分离、计数的影响,从而建立了海水蛭弧菌分离和纯化的方法。应用无营养盐的海水双层琼脂平板法,采用6种宿主菌分别对海南4份海水样品进行计数检测,发现不同宿主菌所得到的蛭弧菌数量不同,同时对分离纯化到的3株海水蛭弧菌进行液体培养增殖,进一步检验了海水双层琼脂法的可靠性。