Basic physico-chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss)

The aim of the study was to compare the physico‐chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). Milt was collected by stripping and spermatozoa concentrations, were determined and compared with sperm motility and spermatocrit values along with seminal plasma indices (pH, osmolality, sodium, potassium, chlorine, calcium, magnesium, glucose and protein concentrations). The highest spermatozoa concentration of 22.3 ± 6.7 × 10⁹ ml^?1 was found in the sea trout milt, and was significantly different of those observed in brook trout (11.9 ± 3.3 × 10⁹ ml^?1) and rainbow trout (10.7 ± 4.4 × 10⁹ ml^?1). The values for pH and K⁺ did not differ significantly among species. The mean pH was 8.0 in the milt of each species and the K⁺ concentrations ranged from 24.8 ± 7.2 to 30.5 ± 7.6 mm L^?1. Considerable differences were determined for the Ca²⁺ ions concentrations. The highest value was found in sea trout (1.7 ± 0.3 mm L^?1), while in the rainbow trout it was 0.7 ± 0.5 and in the brook trout 0.4 ± 0.1 mm L^?1. The most pronounced differences were found in the glucose concentration cause of its unnaturally low concentration in rainbow trout of the mean value of 6.0 ± 15.2 mg L^?1. The mean value in sea trout and brook trout was 185.0 ± 172.4 and 231.2 ± 148.4 mg L^?1 respectively. For all species, protein mean values were below 1.3 g L^?1. The mean osmolality was between 230.6 ± 98.6 and 272.0 ± 26.4 mOsm kg^?1 in the species studied. No correlation was found between any components determined in milt and the spermatozoa motility (P > 0.05). The sperm concentration was positively correlated with the protein content in the milt of the three species studied, other less exhibited correlation was found.     

Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout

The effect of feeding the probiotic Kocuria SM1 to rainbow trout (Oncorhynchus mykiss, Walbaum) on disease resistance was evaluated. Thus, rainbow trout were fed Kocuria SM1 supplemented diets at concentrations of 10⁸ cells g⁻¹ feed for up to four weeks, and then challenged intraperitoneally with Vibrio anguillarum at weekly intervals. A two-week feeding regime led to the maximum reduction in mortalities, i.e. 16%, compared to mortalities of 62, 30 and 22% for one, three and four week feeding regimes, respectively. These compared to 70–90% mortalities of the controls. An enhanced cellular and humoral immune response, notably greater head kidney macrophage phagocytic and peroxidase activities, and higher serum lysozyme and total protein levels were recorded after two weeks of probiotic administration. These results reveal that a two-week feeding regime with Kocuria SM1 leads to higher disease protection in rainbow trout, with protection linked to stimulation of immune parameters.     

Effects of administration of somatostatin-14 and immunoneutralization of somatostatin on endocrine and growth responses in rainbow trout

Injection of somatostatin‐14 (SS‐14) at 5 ng g^?1 body mass (BM) into rainbow trout Oncorhynchus mykiss decreased (P < 0·05, cubic, r² = 0·54) levels of growth hormone (GH) (1·5 ± 0·9 ng ml^?1v. 6·6 ± 0·6 ng ml^?1) over time when compared to controls. Somatostatin‐14 at 50 ng g^?1 BM also decreased (P = 0·064, quadratic; r² = 0·30) levels of GH (3·6 ± 2·1 ng ml^?1v. 6·6 ± 0·6 ng ml^?1) over time compared to controls. In a second study, passive immunization against SS‐14 (1 : 25 dose) increased (P = 0·10, cubic, r² = 0·12) levels of GH (11·0 ± 4·8 ng ml^?1v. 5·2 ± 1·4 ng ml^?1) over time. Passively immunizing against SS‐14 (1 : 50 dose) increased (P < 0·05, cubic, r² = 0·10) levels of GH (8·2 ± 2·3 ng ml^?1v. 5·2 ± 1·4 ng ml^?1) over time compared to controls. Overall, in the active immunization study there was no difference (P > 0·10) in specific growth rate (G) or feed conversion ratio (FCR) between the three treatment groups during the 9 weeks of the study. Only four of the fish immunized against SS‐14, however, developed antibody titres against SS. Compared to controls, these fish exhibited a G of 0·89 ± 0·09 v. 0·56 ± 0·09% per 3 weeks and FCR of 0·80 ± 0·04 v. 1·20 ± 0·05 g g^?1. In SS‐14 immunized fish, levels of GH decreased (P < 0·05) by day 63 while levels of insulin like growth factor‐I (IGF‐I) increased (P < 0·05) by day 42 and 63. These results indicate the hypothalamic hormone SS‐14 regulates GH secretion similarly in rainbow trout as it does in mammals. Active immunization against SS‐14 could improve growth performance in rainbow trout but enhanced G and FCR is dependent upon generation of antibody titres.     

Properties of Probiotics <Emphasis Type="Italic">Kocuria</Emphasis> SM1 and <Emphasis Type="Italic">Rhodococcus</Emphasis> SM2 Isolated from Fish Guts

This study characterized probiotics Kocuria SM1 and Rhodococcus SM2, which were recovered from the intestinal microbiota of rainbow trout (Oncorhynchus mykiss, Walbaum). The cultures were Gram-positive, non-motile, catalase-positive and oxidase-negative cocci or rods. Cell multiplication of SM1 and SM2 was observed at 4–37 °C (45 °C for SM1), in 0–20% (w/v) NaCl and at pH 2–11. The viability was not affected when exposed to pepsin at pH 2.0 and 3.0, and pancreatin at pH 8.0. Neither isolates were chrome azurol S-positive for siderophore production. Of the 19 common enzymes analysed using the API-ZYM system, only 8 were evident in the culture of SM1 compared to 11 enzymes for SM2. The secondary metabolites of both probiotics were inhibitory to Acinetobacter baumannii, Vibrio anguillarum and V. ordalii; SM2 inhibited Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. SM2 was resistant to penicillin and sulphatriad, out of six antimicrobial agents; SM1 was resistant to sulphatriad. These results suggest that Kocuria SM1 and Rhodococcus SM2 are able to grow over a wide range of temperature, salinity and pH, including in conditions that mimic the gastrointestinal environment of fish and produce extracellular enzymes that may have a role in the host digestive processes. Importantly, Rhodococcus SM2 displays a high degree of bacteriocinogenic potential against multi-drug-resistant human pathogens that have never been documented among the gut microbiota of fish.     

Effect of short‐term (0–72 h) fasting on serum biochemical characteristics in rainbow trout Oncorhynchus mykiss

An experiment was carried out to investigate the effects of short‐term fasting periods on the serum biochemical characteristics of rainbow trout, Oncorhynchus mykiss. For this purpose, fish were fasted 0, 6, 12, 24, 48 and 72 h before blood sampling. Thereafter the serum levels of thyroxine (T₄), 3,5,3′‐triiodothyronine (T₃), cortisol, glucose, lactate, triglyceride, cholesterol, total protein, albumin, globulin and albumin : globulin ration (A : G) were determined. Results show that serum levels of T₄ (4.60–8.77 ng ml^?1), T₃ (7.50–13.3 ng ml^?1), cortisol (7.91–24.5 ng ml^?1), glucose (18.5–80.1 mg dl^?1), lactate (12.7–29.6 mg dl^?1), triglyceride (171–500 mg dl^?1), and cholesterol (321–535 mg dl^?1) were significantly affected by the fasting period. However, there were no significant changes in serum total protein (3.03–3.68 g dl^?1), albumin (1.78–2.01 g dl^?1), globulin (1.15–1.70 g dl^?1) or A : G (1.13–1.93) among the fish fasted 0–72 h. Results clearly show the importance of a fasting period on the serum biochemical properties of rainbow trout. According to the results, 24 h fasting is suggested as a pre‐sampling fasting period in rainbow trout to measure serum levels of T₄, T₃, cortisol, glucose, lactate, triglyceride, and cholesterol. Potential mechanisms related to the changes in biochemical properties are discussed.     

Thermal tolerance and metabolic physiology among redband trout populations in south‐eastern Oregon

Streamside measurements of critical thermal maxima (T_crit), swimming performance (U_crit), and routine (R_r) and maximum (R_max) metabolic rates were performed on three populations of genetically distinct redband trout Oncorhynchus mykiss in the high‐desert region of south‐eastern Oregon. The T_crit values (29·4 ± 0·1° C) for small (40–140 g) redband trout from the three streams, and large (400–1400 g) redband trout at Bridge Creek were not different, and were comparable to published values for other salmonids. At high water temperatures (24–28° C), large fish incurred higher metabolic costs and were more thermally sensitive than small fish. U_crit(3·6 ± 0·1 L_F s^?1), R_r(200 ± 13 mg O₂ kg^?0·830 h^?1) and metabolic power (533 ± 22 mg O₂ kg^?0·882 h^?1) were not significantly different between populations of small redband trout at 24° C. R_max and metabolic power, however, were higher than previous measurements for rainbow trout at these temperatures. Fish from Bridge Creek had a 30% lower minimum total cost of transport (C_min), exhibited a lower refusal rate, and had smaller hearts than fish at 12‐mile or Rock Creeks. In contrast, no differences in U_crit or metabolism were observed between the two size classes of redband trout, although C_min was significantly lower for large fish at all swimming speeds. Biochemical analyses revealed that fish from 12‐mile Creek, which had the highest refusal rate (36%), were moderately hyperkalemic and had substantially lower circulating levels of free fatty acids, triglycerides and albumin. Aerobic and anaerobic enzyme activities in axial white muscle, however, were not different between populations, and morphological features were similar. Results of this study: 1) suggest that the physiological mechanisms that determine T_crit in salmonids are highly conserved; 2) show that adult (large) redband trout are more susceptible to the negative affects of elevated temperatures than small redband trout; 3) demonstrate that swimming efficiency can vary considerably between redband trout populations; 4) suggest that metabolic energy stores correlate positively with swimming behaviour of redband trout at high water temperatures; 5) question the use of T_crit for assessing physiological function and defining thermal habitat requirements of stream‐dwelling salmonids like the redband trout.     

The effects of environmental salinity on the growth and physiology of totoaba Totoaba macdonaldi and shortfin corvina Cynoscion parvipinnis

Totoaba Totoaba macdonaldi and shortfin corvina Cynoscion parvipinnis, were acclimated and reared together at salinities of 0, 2, 5, 10, 20 and 35 for 56 days. Initial overall mean ± s.d . body masses of 67·6 ± 7·1 g T. macdonaldi and 37·3 ± 3·1 g C. parvipinnis increased to final overall masses of 217·4 ± 30·3 and 96·5 ± 16·5 g, respectively, at the end of the study. Totoaba macdonaldi was not able to tolerate salinities of 0 and 2 and C. parvipinnis of 0. In contrast, both species had 100% survival at salinities ≥ 10. Somatic growth was highest not at natural seawater salinity of 35, but at 10. Plasma osmolality ranged from 172·5 to 417·0 mOsmol kg^?1 for T. macdonaldi and from 207·0 to 439·5 mOsmol kg^?1 for C. parvipinnis and varied in direct proportion to salinity. The estimated isosmotic salinities of T. macdonaldi and C. parvipinnis were 12·3 and 13·4, respectively. Cynoscion parvipinnis reared at two had significantly lower plasma lysozyme activity (95·0 Units ml^?1) than fish held at salinities from 5 to 35 (ranging from 215·0 to 355·0 Units ml^?1), but without clear trends over this range. Blood neutrophil oxidative radical production (NBT) (ranging from 3·9 to 6·7 mg ml^?1) had some significant differences among salinities, but these did not follow a clear pattern. For T. macdonaldi, neither lysozyme activity nor NBT was affected by salinity. Ash content of whole fishes varied directly and moisture content inversely, with salinity for both species.     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.     

Respiratory-cardiovascular physiology and chloroethane gill flux in the channel catfish, Ictalurus punctatus

A fish respirometer-metabolism chamber was used to obtain in vivo respiratory-cardiovascular and chloroethane gill flux data on transected channel catfish (Ictalurus punctatus). Methods used for spinal transection, attachment of an oral membrane (respiratory mast), placement and attachment of blood cannulas and urine catheters are described. Respiratory physiology, cardiac output and chemical extraction efficiencies for 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) were determined on 419–990 g catfish. The overall mean values (± s.d.) for ventilation volume (Q_v), effective respiratory volume (Q_w), oxygen consumption (Vo₂ and percentage utilization of oxygen (U) were 17-3 ±4–71 h^?1 kg^?1, 9·8±l·71 h^?1 kg^?1, 71·6±12·5mg h^?1 kg^?1, and 49± 10%, respectively, while cardiac output calculated via the Fick Method was 2·4±0·61 h^?1 kg^?1. Additional measurements were made on ventilation rate (V_r), total plasma protein, haematocrit (Hct), and urine volume; while both arterial and venous blood were analysed for pH, oxygen partial pressure (P02), carbon dioxide partial pressure (Pco₂), total oxygen (To₂), total carbon dioxide (Tco₂) and total ammonia (TAMM). Physiological measurements taken at 24 h were not significantly different from those taken at 48 h and indicated no deterioration of the in vivo preparation. All of these values agreed well with literature values on UTitransected channel catfish, except for Hct which was lower for cannulated animals used in this study. Overall, these data provide strong support for the use of transected channel catfish for in vivo collection of physiological and chemical gill flux data. The mean initial chemical extraction efficiencies for TCE, PCE and HCE were 41, 61 and 73%, respectively. Chemical clearances (Cl_X) for these same three chemicals were 5·9, 9·3 and 10·8 1 h^?1 kg^?1, respectively. The approximate 1: 1 relationship between effective respiratory volume (Q_w) and chemical clearance (Cl_x) indicated that branchial uptake of PCE and HCE was water flow-limited. Chemical gill flux observed for channel catfish and chloroethanes was similar to that observed for rainbow trout in previous studies and provided further support for the flow-limited model of chemical flux across fish gills.     

Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa

Aims: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi‐resistant) Pseudomonas aeruginosa. Methods and Results: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N‐phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10⁶Ps. aeruginosa cells ml^?1 in presence of 10 mmol l^?1 ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml^?1 endolysin EL188 led to a strain‐dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml^?1 further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). Conclusions: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. Significance and Impact of the Study: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as ‘enzybiotics’ must not be limited to gram‐positive pathogens.     

Content of cysteine, reduced and oxidized glutathione in spermatozoa of representatives of Acipenseriformes (Acipenser baerii and A. ruthenus) as well as teleosts (Perca fluviatilis and Sander lucioperca)

Glutathione belongs to a vital intra‐ and extra‐cellular protective antioxidant and is found almost exclusively in its reduced form. The ratio between its reduced and oxidized within cells is often used as a marker of cellular toxicity. The objectives of the study were to (i) determine both the reduced (GSH) and oxidized glutathione (GSSG) and cysteine (Cys) in the sperm of the Acipenser baerii and Acipenser ruthenus, as well as in Perca fluviatilis and Sander lucioperca, and (ii) to demonstrate the differences in concentration levels between representatives of acipenseriform and teleost species. High performance liquid chromatography with electrochemical detection was employed. The average content of the thiols determined in the sperm samples were as follows: Acipenser baerii (cysteine 55 ± 8 μg ml^?1; GSH 126 ± 19 μg ml^?1; GSSG 49 ± 7 μg ml^?1), Acipenser ruthenus (cysteine 62 ± 9 μg ml^?1; GSH 768 ± 115 μg ml^?1; GSSG 180 ± 16 μg ml^?1), Sander lucioperca (cysteine 251 ± 38 μg ml^?1; GSH 185 ± 28 μg ml^?1; GSSG 93 ± 14 μg ml^?1), Perca fluviatilis (cysteine 281 ± 42 μg ml^?1; GSH 496 ± 74 μg ml^?1; GSSG 138 ± 21 μg ml^?1). Based on the results obtained it can be concluded that this method is sensitive and selective for the determination of these compounds in real samples. Results revealed differences in cysteine content between species of the two systematic categories but also showed that ratios between GSH and GSSG can vary between species while potentially predict oxidative stress in fish sperm.     

Fine structure and motility of spermatozoa and composition of the seminal plasma in the perch

The fine structure and motility of spermatozoa and the composition of the seminal plasma of the perch Perca fluviatilis are investigated by electron microscopy, computer assisted cell motility analysis (CMA) and biochemical methods. The spermatozoon is asymmetrical as the flagellum inserts mediolateral on the nucleus. It lacks an acrosome, has an ovoid head and a small midpiece with one mitochondrion. Sperm motility–initiated in distilled water (10° C)–is characterized as follows: 85·0 ± 2·7% of the spermatozoa are motile, the main swimming type (10 ± 1 s after motility initiation) is the linear motion (61·4 ± 24·4%) and the average swimming velocity is 122·4 ± 21·9 μm s^–1. When motility is initiated with NaCl, glucose or sucrose solutions of 100 mosmol kg^–1 the percentage of motile spermatozoa and the swimming types are similar as in water, but the swimming velocity (174·0 ± 22·3 μm s^–1) is significantly higher. Motility is inhibited by high osmolality of the diluent: when increasing the osmolality of the saline solutions to 350 mosmol kg^–1 sperm motility is totally suppressed while potassium (10–40 mmol 1^–1) does not affect motility parameters. pH optimum for sperm motility is between pH 7·0 and 8·5. The seminal fluid contains 124·01 ± 21·68 mmol 1^–1 sodium, 10·22 ± 1·11 mmol 1^–1 potassium and 0·72 ± 0·26 mmol 1^–1 calcium. pH is 8·25 ± 0·09, and osmolality 283·90 ± 37·19 mosmol kg^–1. The following organic components were determined: monosaccharides (glucose 63 ± 19 μmol 1^–1, fructose 54 ± 28 μmol 1^–1, galactose 59 ± 25 μmol 1^–1), lipids (cholesterol 5·51 ± 6·42 μmol 1^–1, triglycerides 72 ± l00 μmol l^–1, cholesteryloleate 15–150 μmol 1^–1, phosphatidylcholine 26 · 31 μmol 1^–1, glycolipids 1–10 mg 100 m1^–1), lactate 108 ± 99 μmol 1^–1, hydroxybutyrate 102 ± 99 nmol 1^–1, choline 59 ± 159 μmol 1^–1, protein 344·75 ± 59·06 mg 100m1^–1, enzymes (β-d -glucuronidase l.4 ± 0.7 μmol h^–1 100 ml^–1, protease (caseolytic activity) 1·0 ± 0·6 μmol h^–1 100 ml^–1, alkaline phosphatase 2520·0 ± 861·0 μmol h^–1 100 ml^–1, acid phosphatase 44.0 ± 16.0 μmol h^–1 100 ml^–1, glucose-6-phosphate dehydrogenase 38·9 ± 86·9 μmol h^–1 100 ml^–1, lactate dehydrogenase 134·4 ± 69·6 μmol h^–1 100 ml^–1, butyrylcholine esterase 0·014 ± 0·010 μmol h^–1 100 ml^–1, adenosine triphosphatase 562·8 ± 665·4 μmol h ^–1 100 ml^–1).     

Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum‐contaminated soil

Aims: To isolate and characterize the biosurfactant‐producing micro‐organism from petroleum‐contaminated soil as well as to determine the biochemical properties of the biosurfactant. Methods and Results: A novel rhamnolipid‐producing Pseudomonas aeruginosa (GenBank accession number GQ241355 ) strain was isolated from a petroleum‐contaminated soil. Surface active compound was separated by solvent extraction of the acidified culture supernatant. The extract was able to reduce the surface tension of water from 72 to 44 mN m^?1 at a critical micelle concentration of 11·27 ± 1·85 mg l^?1. It showed better activity (based on microdilution method) against Gram‐positive (≤ 31 mg ml^?1) bacteria and filamentous fungi (≤ 50 mg ml^?1) than Gram‐negative bacteria (≥ 125 mg ml^?1) with mild toxicity (HC₅₀– 38 ± 8·22 μg ml^?1) to red blood cells. Fourier transform infrared spectroscopy revealed the presence of aliphatic chain, hydroxyl groups, ester and glycosidic bonds. Presence of nineteen rhamnolipid homologues with variation in chain length and saturation was revealed from liquid chromatography coupled to mass spectrometry with electrospray ionization. Conclusion: The results indicate that the isolated biosurfactant has a novel combination of rhamnolipid congeners with unique properties. Significance and Impact of the Study: This study provides a biosurfactant, which can be used as a biocontrol agent against phytopathogens (Fusarium proliferatum NCIM 1105 and Aspergillus niger NCIM 596) and exploited for biomedical applications.     

Chronic stress of rainbow trout Oncorhynchus mykiss at high altitude: a field study

The stress response of Oncorhynchus mykiss in high‐altitude farms in central Mexico was investigated over two seasons: the cool (9·1–13·7° C) dry winter season, and the warmer (14·7–15·9° C), wetter summer season. Fish were subjected to an acute stress test followed by sampling of six physiological variables: blood cortisol, glucose, lactate, total antioxidant capacity, haemoglobin concentration and per cent packed cell volume (V_PC%). Multivariate analyses revealed that lactate and total antioxidant capacity were significantly higher in the summer, when water temperatures were warmer and moderate hypoxia (4·9–5·3 mg l^?1) prevailed. In contrast, plasma cortisol was significantly higher in the winter (mean ± s.e .: 76·7 ± 4·0 ng ml^?1) when temperatures were cooler and dissolved oxygen levels higher (6·05–7·9 mg l^?1), than in the summer (22·7 ± 3·8 ng ml^?1). Haemoglobin concentrations (mg dl^?1) were not significantly different between seasons, but V_PC% was significantly higher in the summer (50%) than in the winter (35%). These results suggest that in summer, effects of high altitude on farmed fish are exacerbated by stresses of high temperatures and hypoxia, resulting in higher blood lactate, increased total antioxidant capacity and elevated V_PC% levels.     

Development of a microtitre plate indirect ELISA for measuring cortisol in teleosts, and evaluation of stress responses in rainbow trout and gilthead sea bream

A microtitre plate indirect enzyme‐linked immunoassay (ELISA) was developed for measuring plasma cortisol levels in rainbow trout Oncorhynchus mykiss, gilthead sea bream Sparus auratus sea bass Dicentrarchus labrax and Senegalese sole Solea senegalensis. Covalink microplates pretreated with disuccinimidyl suberate were coated with bovine serum albumin (BSA) conjugated to cortisol‐3‐carboxymethyl oxime. After blocking with BSA, competition was started by addition of plasma samples and anti‐cortisol antibody raised in rabbit. Goat anti‐rabbit IgG conjugated‐peroxidase was added as second antibody and then incubated with orthophenylenediamine as substrate. Reaction was stopped with 0·1 M HCl and absorbance was read at 450 nm in an automatic plate reader. The standard curve was linear from the lower limit of sensitivity of the assay (c. 0·3 ng ml^?1) to c. 3000 ng ml^?1. Dose‐response inhibition curves using serially diluted plasma samples of four species consistently showed parallelism with the standard curve using cortisol. The ELISA satisfied the strictest criteria of specificity (cross‐reactivity of anti‐cortisol antibody with testosterone, progesterone and 17ß‐oestradiol was negligible, cross‐reactivity with cortisone, corticosterone and 11‐deoxycortisol, was 1·5, 1 and 0·1%, respectively), reproducibility (interassay CV <6%), precision (intra‐assay CV <4%), and accuracy (average recovery >98%). Plasma cortisol concentration in rested fishes was in the range of 5–30 ng ml^?1. To physiologically validate the technique, changes in plasma cortisol concentrations were also measured in plasma of rainbow trout and gilthead sea bream following an acute 15 min chasing or 3 min air‐exposure stress, respectively. In both species plasma concentrations of cortisol, glucose and lactate rose significantly with respect to controls, showing concentrations similar to those reported previously for these species under similar stress conditions. Furthermore, gilthead sea bream chronically stressed by maintaining for 14 days under increased stocking density conditions also showed increased concentrations of plasma cortisol and glucose. These results validate the indirect ELISA technique developed for use in the evaluation of plasma cortisol concentration of at least four fish species.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Evaluation of waste chicken feathers as peptone source for bacterial growth

Aims: Peptones are one of the most expensive constituents of microbial media. This study was undertaken to prepare the peptone from waste chicken feathers through a new process. Methods and Results: The chemical analysis of chicken feather peptone (CFP) was performed. The ability of CFP to support the growth of the three test bacteria in liquid and agar media was comparable to those of three commercial peptones [tryptone peptone (TP), fish peptone and protease peptone (PP)]. Conclusions: CFP was found to be rich in ash (42·1 g 100 g^?1), protein (55·8 g 100 g^?1) and mineral contents. The maximum biomass yield (3·13 g l^?1) and colony number (83 × 10⁸ CFU ml^?1) for bacterium Bacillus subtilis were attained with CFP. The maximum biomass yields and colony numbers for Lactobacillus delbrueckii ssp. bulgaricus and Escherichia coli were reached in TP medium. Second high biomass yield (2·64 g l^?1) and colony number (75 × 10⁸ CFU ml^?1) for E. coli were achieved using CFP. Third high biomass yield (1·29 g l^?1) and colony number (90 × 10⁷ CFU ml^?1) for Lact. delbrueckii ssp. bulgaricus were obtained in CFP medium. Significance and Impact of the Study: Usability of waste chicken feathers as substrate for bacteria was investigated for the first time in the present study. The peptone may be used in industrial fermentations for production of antibiotics, organic acids, enzymes and biopolymer. It may be also used in clinical microbiology. A new chemical process was developed for peptone preparation. This process may be also employed for peptone preparation from other organic materials, especially fibrose protein‐containing materials.     

The reproductive cycle of female Ballan wrasse Labrus bergylta in high latitude,temperate waters

This 2 year study examined the reproductive cycle of wild female Ballan wrasse Labrus bergylta in western Norway as a precursor to captive breeding trials. Light microscopy of ovarian histology was used to stage gonad maturity and enzyme‐linked immuno‐absorbent assay (ELISA) to measure plasma concentrations of the sex steroids testosterone (T) and 17β‐oestradiol (E₂). Ovarian recrudescence began in late autumn to early winter with the growth of previtellogenic oocytes and the formation of cortical alveoli. Vitellogenic oocytes developed from January to June and ovaries containing postovulatory follicles (POF) were present between May and June. These POF occurred simultaneously among other late maturity stage oocytes. Plasma steroid concentration and organo‐somatic indices increased over winter and spring. Maximal (mean ±s.e .) values of plasma T (0·95 ± 0·26 ng ml^?1), E₂ (1·75 ± 0·43 ng ml^?1) and gonado‐somatic index (I_G; 10·71 ± 0·81) occurred in April and May and decreased greatly in July when only postspawned fish with atretic ovaries occurred. Evidence indicates that L. bergylta are group‐synchronous multiple spawners with spawning occurring in spring and peaking in May. A short resting period may occur between late summer and autumn when previtellogenic oocytes predominate and steroid levels are minimal.     

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

Aims: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Methods and Results: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar–Hinton broth supplemented with 0·25 μmol DIC or thymol ml^?1 but not with 0·01 μmol monensin ml^?1 or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log₁₀ CFU from controls after 24 h pure culture in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Similarly, each test Campylobacter strain was reduced >3 log₁₀ CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Treatments with 0·25 μmol thymol ml^?1, 0·01 μmol monensin ml^?1 or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0·25 or 1·0 μmol DIC ml^?1 but only intermittently reduced, if at all, by the other treatments. Conclusions: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Significance and Impact of the Study: Results suggest a potential physiological characteristic that may be exploited to develop interventions.     

Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting

Aims:  Zero‐valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. Methods:  A field‐scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c. 8·5 log CFU 100 ml^?1 of Escherichia coli O157:H12 was introduced to all three column treatments in 20‐l doses. Filtered waters were subsequently overhead irrigated to ‘Tyee’ spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. Results:  ZVI filters inactivated c. 6 log CFU 100 ml^?1E. coli O157:H12 during filtration on day 0, significantly (P < 0·05) more than S filter (0·49 CFU 100 ml^?1) when compared to control on day 0 (8·3 log CFU 100 ml^?1). On day 0, spinach plants irrigated with ZVI‐filtered water had significantly lower E. coli O157 counts (0·13 log CFU g^?1) than spinach irrigated with either S‐filtered (4·37 log CFU g^?1) or control (5·23 log CFU g^?1) water. Soils irrigated with ZVI‐filtered water contained E. coli O157:H12 populations below the detection limit (2 log CFU g^?1), while those irrigated with S‐filtered water (3·56 log CFU g^?1) were significantly lower than those irrigated with control (4·64 log CFU g^?1). Conclusions:  ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. Significance and Impact of the Study:  Zero‐valent ion treatment may be a cost‐effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.