Effect of dietary administration of Porphyridium cruentum on the respiratory burst activity of sole, Solea senegalensis (Kaup), phagocytes

The stimulatory effect of the red microalga Porphyridium cruentum on respiratory burst activity of sole phagocytes was evaluated in vivo . Oral administration of a diet supplemented with lyophilized P. cruentum cells (10 g kg⁻¹) stimulated respiratory burst activity after 4 weeks feeding in sole vaccinated with Photobacterium damselae subsp. piscicida bacterin.     

The Culture of the Rotifer Brachionus plicatilis with Chlorella vulgaris Containing Vitamin B12 in its Cells

The rotifer Brachionus plicatilis requires vitamin B₁₂. Freshwater Chlorella , which is produced by traditional culture, cannot support rotifer growth under bacteria-free conditions. However, Chlorella enriched with vitamin B₁₂ can support rotifer growth. To attain stable mass production of Brachionus , it is desirable to develop a food that can completely support growth of the rotifers. The authors cultured rotifers at experimental and mass culture scale with concentrated Chlorella vulgaris suspension enriched with vitamin B₁₂ in their cells.
Chlorella suspensions were prepared containing different amounts of vitamin B₁₂ in their cells, and rotifers were then cultured in 20 ml of the prepared suspensions. The highest rotifer yield was obtained from the group cultured with Chlorella containing more vitamin B₁₂ in their cells. The suitable content of vitamin B₁₂ in the concentrated Chlorella suspension commercially available for mass culture of the rotifer is considered to be 200 _μg per 100 g dry matter of Chlorella . The amount of vitamin B₁₂ necessary to produce one individual rotifer is calculated at 0.32 pg.
The authors conducted mass production of the rotifer with baker's yeast and refrigerated concentrated Chlorella containing vitamin B₁₂ Rotifer culture with vitamin B₁₂ was more stable and showed 1.3 times higher production than with normal Chlorella .     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae , being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD₅₀ values ranging from 3.9 × 10³ to 1.5 × 10⁸ cfu at 20 °C. The virulence was significantly higher at 20 °C than at 13 °C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.     

Random sequencing of genomic DNA of <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>

ABSTRACT:   Random sequencing of the genomic DNA of Photobacterium damselae subsp. piscicida was conducted. The sequences were assembled into 930 contigs. The total length of these contigs accounts for 37.5% of the genome of P. damselae subsp. piscicida. The contigs contain 2055 open reading frames (ORFs) that have homology with genes in the GenBank database. Furthermore, some of the ORFs have homology with reported virulence related genes, and are classified as encoding colonization factors, exotoxin, and lipopolysaccharide (LPS). Thirty-seven ORFs have homology with colonization factors. In those colonization factors, 27, three, and four ORFs have homology with polar flagellar-related genes, capsule-related genes, and others (accessory colonization factors, superoxide dismutase (Cu-Zn), MshA biogenesis protein, and heme receptor genes), respectively. Five ORFs have homology with exotoxin-related genes (2 hemolysin, phospholipase, hyaluronidase, lysophospholipase L2 genes). Further, six ORFs have homology with LPS-related genes.     

Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.     

Selenium nutrition of hybrid striped bass (Morone chrysops × M. saxatilis) bioavailability, toxicity and interaction with vitamin E

Two concurrent 12-week feeding trials were conducted to evaluate the bioavailability of inorganic sodium selenite and organic seleno-DL-methionine and to investigate the potential interaction between selenium and vitamin E in juvenile hybrid striped bass. In experiment 1, purified diets utilizing casein, gelatin and an amino acid premix as protein sources with a basal selenium concentration of 0.11 mg Se   kg⁻¹ were supplemented with either Na₂SeO₃ to provide selenium concentrations of 1.19, 2.00, 5.17 and 21.23 mg Se kg⁻¹ or with seleno-DL-methionine to provide 0.90, 1.26 and 2.55 mg Se kg⁻¹ and fed to juvenile hybrid striped bass in aquaria. A second experiment evaluated potential interactions by feeding these purified diets with or without supplemental vitamin E or sodium selenite, singularly or in combination. No overt selenium deficiency signs were exhibited by fish in either of the experiments; however, signs of selenium toxicity including retarded weight gain (WG), reduced feed intake and feed efficiency ratio (FER) as well as increased mortality, were observed in fish fed the diet containing more than 20 mg Se kg⁻¹. Whole-body selenium and whole-body selenium retention were linearly influenced by sodium selenite and selenomethionine. However, there was no significant effect of dietary selenium, vitamin E or their interaction on WG, FER and survival. Slope-ratio analysis showed that bioavailability of seleno-DL-methionine as a selenium source for juvenile hybrid striped bass was significantly ( P  < 0.01) higher (3.3-fold) than sodium selenite.     

Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor gene

A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor.     

Adhesion to sole, Solea senegalensis Kaup, mucus of microorganisms isolated from farmed fish, and their interaction with Photobacterium damselae subsp. piscicida

Abstract Most studies carried out to select microorganisms as candidate probiotics have focused on in vitro antagonism tests, such as the production of inhibitory compounds against pathogenic microorganisms. However, attachment to mucous surfaces could be another criterion to be considered when selecting potential probiotics for aquaculture. Nineteen isolates obtained from farmed Senegalese sole, Solea senegalensis Kaup, and gilthead sea bream, Sparus aurata L., have been evaluated for their capacity to adhere to skin and intestinal mucus of Senegalese sole, and their antagonistic effect against Photobacterium damselae subsp. piscicida, an important pathogen for farmed sole. The isolates from gilthead sea bream showed the highest percentage of adhesion to sole mucus, whilst the pathogenic microorganisms assayed and the isolates from sole showed, in general, a lower ability to adhere to sole mucus. The results suggest that the adhesion to fish mucus was more dependent on the isolate tested than on the host mucus. The isolates from gilthead sea bream also showed a higher antagonistic activity against P. damselae subsp. piscicida than those from Senegalese sole. Four isolates were selected, on the basis of their adhesive ability and antagonistic effect on P. damselae subsp. piscicida, to study their interactions with the pathogen in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions. The results obtained show the ability of three isolates to reduce the adhesion of P. damselae subsp. piscicida to sole mucus under displacement and competition conditions. The adhesion of the pathogen to sole intestinal mucus was also significantly reduced when three isolates were assayed under exclusion conditions.     

Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.     

Effect of Dietary Supplementation of Vitamins A, D3, E, and C on Yearling Rice Field Eel, Monopterus albus: Serum Indices, Gonad Development, and Metabolism of Calcium and Phosphorus

Abstract.— The objective of this study was to determine the effect of dietary vitamins A, D₃, E, and C on the gonad development, lipid peroxidation, and immune response of yearling rice field eel, Monopterus albus . A 6-wk feeding trial was designed according to an L₁₆(4⁵) orthogonal design, in which four vitamins, each at four supplementation levels, were arranged. Sixteen diets were mixed with the different vitamin levels and randomly assigned to 16 groups of fish. Increasing dietary vitamin E supplementation level significantly ( P  ≤ 0.05) increased the gonadosomatic index and lowered the serum content of malondialdehyde of rice field eel. Increasing dietary vitamin A and C levels also showed similar effect, but the differences were not statistically significant. Serum immunoglobulin M content increased significantly ( P  ≤ 0.01) as dietary vitamin C supplementation levels increased. The concentrations of calcium in bones showed significant ( P  ≤ 0.05) trend with vitamin D₃ and A supplementation levels, but the bone phosphorus content was not affected by the dietary vitamin levels.     

Cloning and characterization of Photobacterium damselae ssp. piscicida phospholipase: an enzyme that shows haemolytic activity

A phospholipase gene of Photobacterium damselae ssp. piscicida (ppp) was cloned from a genomic library and its nucleotide sequence was determined. The open reading frame consisted of 1218 bp encoding a protein of 405 amino acids with a predicted molecular mass of 46 kDa. The PPP had identities (53-55%) with phospholipase and haemolysin of Vibrio spp., while it showed low identities (23-26%) with glycerophospholipid cholesterol acyltransferase of Aeromonas spp. A recombinant PPP (rPPP) with a His tag at the C-terminus expressed in Escherichia coli and purified showed phospholipase activity. The rPPP also showed lecithin-dependent haemolytic activity against mammalian erythrocytes and direct haemolytic activity against fish erythrocytes. The culture supernatant of wild-type P. damselae ssp. piscicida showed phospholipase activity, while that of a PPP gene knockout mutant did not.     

Evaluation of carbohydrate rich diets through common carp culture in manured tanks

Four diets (T₀–T₃) were formulated reducing the fishmeal (Indian) component by 100 g kg^–1 from 300 to 0 g kg^–1 and including proportionately increasing quantities of maize. Diets were fed for 120 days at 50 g kg^–1 body weight to triplicate groups of common carp (av. wt. 2.11–2.18 g) stocked at 1 m^–2 in mud bottomed cement tanks (18 m²), fertilized with poultry manure. Fish growth, SGR and FCR in the different treatments were statistically not significantly different ( P  > 0.05). PER was lowest for the 300 g fishmeal kg^–1 diet treatment (diet T₀), increasing with decrease in dietary fishmeal content (diets T₁–T₃). Fish survival ranged from 96.29 to 100%. Diets influenced carcass composition and digestive enzyme activity. A significant increase in lipid deposition was recorded with increasing dietary carbohydrate content. Amylase, protease and lipase activities were higher in fish fed with diets T₂ and T₃. The protein sparing effect of dietary carbohydrate and the economic implication of eliminating fishmeal from the diet are discussed.     

Selection of probiotic bacteria for use in shrimp larviculture

Three candidate probiotics, Bacillus foraminis, Bacillus cereus biovar toyoi and Bacillus fusiformis , were isolated from hydrogen-producing fermented solution and identified using 16S rRNA sequence analysis. Bacillus foraminis and B. cereus biovar toyoi exhibited strong antagonism against Streptococcus iniae and Photobacterium damselae subsp. piscicida in in vitro co-culture for competitive exclusion assay and then were conducted in the larviculture system of Penaeus monodon reared from zoea 1 to postlarva 1. The daily addition of B. cereus biovar toyoi resulted in significantly deleterious effects on survival ( P <0.01) whereas the daily addition of B. fusiformis showed highest survival rate (88.7±0.7%) but no statistically significant difference from control (73.3±12.1%). Bacillus fusiformis was continuously applied in the larviculture system of Litopenaeus vannamei . Administration of B. fusiformis significantly increased survival ( P <0.01) in both treatments added daily (87.9±1.7%) and every other day (54.7±1.2%), respectively, at a concentration of 10⁵ CFU mL⁻¹ over control (41.2±1.3%).     

Comparative Sensitivity of Yellowtail Seriolu quinqueradiata and Goldstriped Amberjack S. aureovittata to Photobacterium damsela subsp. piscicida

The pathogenicity of Photobacterium damsela subsp. piscicida to yellowtail Seriola quinqueradiata and goldstriped amberjack S. aureovittata was investigated. The 10-d cumulative mortality of yellowtail and goldstriped amberjack challenged by 1.2 × 10² CFU/fish of P. damsela piscicida was 70% and 10%. respectively. Serum bactericidal activity of goldstriped amberjack was higher than that of yellowtail. Although the phagocytic activity against P. damsela piscicida was not observed in kidney leukocytes of yellowtail and goldstriped amberjack, the production of superoxide anion in goldstriped amberjack kidney leukocytes against P. damsela piscicida was higher than that of yellowtail.     

Nutritional evaluation of waste date fruit as partial substitute for soybean meal in practical diets of juvenile Nile tilapia, Oreochromis niloticus L.

The potential of waste date meal (WDM; low-quality date palm, Phoenix dactylifera L.) as a carbohydrate source in formulated diets for Nile tilapia was evaluated. Four isocaloric-practical diets (15.7 kJ g⁻¹) were formulated incorporating WDM at 0, 100, 200 and 300 g kg⁻¹ levels as partial substitutes for soybean meal (SBM). These were designated D₀ [284 g crude protein (CP) and 383 g carbohydrate (CHO) kg⁻¹ diet], D₁ (279 g CP and 446 g CHO kg⁻¹ diet), D₂ (207 g CP and 495 g CHO kg⁻¹ diet) and D₃ (175 g CP and 578 g CHO kg⁻¹ diet). Each diet was fed to three replicate groups of 30 fish [20.20 ± 0.09 g (±SE)] for 75 days. No feed-related mortality was observed during the entire experimental period. Final body weight (FBW) and specific growth rate (SGR) in the different treatments were statistically not significantly different ( P  > 0.05). Protein efficiency rate (PER) was lowest in diet D₀ and increased with decrease of SBM content (D₁–D₃). A significant increase in whole body lipid content was recorded in fish fed diets D₂ and D₃. Results showed that WDM could be a substitute for SBM up to 300 g kg⁻¹ in practical Nile tilapia diets without compromising growth.     

Microbial Phytase Supplementation in Rohu, Labeo rohita, Diets Enhances Growth Performance and Nutrient Digestibility

Abstract.— A feeding trial was conducted for 60 d to study the effect of dietary microbial phytase on growth performance and nutrient digestibility of rohu, Labeo rohita , fingerlings. One hundred and twenty fingerlings (average initial weight: 9.17 g/fish) were equally distributed into five experimental tanks, each with four replicates. Five isonitrogenous (35%) and isocaloric (16.79 kJ/g) diets were prepared from plant-based ingredients, supplemented with microbial phytase at the level of 0, 250, 500, 750, and 1000 U/kg diets and fed to T₀, T₁, T₂, T₃, and T₄ groups, respectively. Weight gain %, food conversion ratio, protein efficiency ratio, and apparent net protein utilization were significantly ( P  < 0.05) improved in groups fed phytase-supplemented diets compared to control, the highest being observed in T₃ group. Maximum apparent digestibility of phosphorus and crude protein was recorded in T₃ group. Bone ash, phosphorus (P), and calcium (Ca) contents were also significantly ( P  < 0.05) increased in phytase-fed groups. However, maximum was recorded in T₃ group. Results from the present study indicated that addition of 750 U microbial phytase/kg diets effectively improved nutrient utilization, bone mineralization, and hence growth of L. rohita fingerlings.     

Elevation of lipid peroxide level and production of hydroxy lipids in cultured Hepa-T1 cells by oxidative stressors

ABSTRACT:   In order to investigate the relationships between levels of lipid peroxides (LPO), hydroxy lipids (L-OH) and of antioxidative enzymes in live fish, cultured cells from tilapia liver (Hepa-T1) were treated with oxidative stressors, 2,2'-azobios(2-amidino-propane) dihydrochloride (AAPH) and H₂O₂. By treating with 5 mM AAPH, LPO and L-OH of the cells significantly increased time-dependently. In particular, L-OH increased from 7.6 ± 0.7 to 23.6 ± 1.8 nmol/mg protein after 2 h of treatment, and synchronously the glutathione peroxidase (GPx) activity of the cells increased from 259 ± 106 to 1970 ± 135 mU/mg protein. In the case of H₂O₂, however, no elevation of L-OH was observed. In this case, catalase (CAT) and superoxide dismutase (SOD) activities increased time-dependently. Especially, after 2 h exposure, the CAT activity increased remarkably from 217 ± 21 to 2510 ± 120 mU/mg protein, though the GPx activity did not change significantly. These results suggest that the elevation of the L-OH level is closely associated with the elevation of GPx activity, but not with that of CAT or SOD. However, when vitamin E-enriched Hepa-T1 cells were treated with AAPH and H₂O₂, the production of LPO and L-OH was suppressed significantly. The findings suggest that vitamin E also plays an important role in the levels of the oxidation products in vivo .     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Dietary vitamin E requirement of hybrid striped bass (Morone chrysops female × M. saxatilis male)

This study was conducted to determine the dietary vitamin E requirement of juvenile hybrid striped bass ( Morone chrysops female ×  Morone saxatilis male). Semi-purified diets supplemented with 0.2 mg Se kg⁻¹ from Na₂SeO₃ and either 0 (basal), 10, 20, 40, 60, or 80 mg vitamin E kg⁻¹ as  DL -α-tocopheryl acetate were fed to hybrid striped bass initially averaging 1.8 ± 0.1 g (mean ± SD) for 12 weeks. Fish fed the basal diet, which contained 5.8 mg α-tocopherol kg⁻¹ dry weight, were darker in colour and had reduced weight gain, as well as generally reduced haematocrit values compared with fish fed diets supplemented with vitamin E. In addition, fish fed diets containing less than 20 mg supplemental vitamin E kg⁻¹ had significantly ( P  < 0.05) reduced weight gain and feed efficiency compared with those fed diets supplemented with vitamin E at 20–80 mg kg⁻¹. Dietary supplementation of vitamin E caused incremental increases in the concentration of α-tocopherol in both plasma and liver tissues. However, hybrid striped bass fed graded levels of vitamin E did not exhibit a dose response in terms of ascorbic acid-stimulated lipid peroxidation of hepatic microsomes. Regression analysis of weight gain data using the broken-line model indicated a minimum vitamin E requirement ( ±  SE) of 28 ( ±  3) mg kg⁻¹ dry diet. Based on these data, the dietary vitamin E requirement of hybrid striped bass appears to be similar to that determined for other fish species.