Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assay's negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.     

Functional Feeds to Tackle Meagre (Argyrosomus regius) Stress: Physiological Responses under Acute Stressful Handling Conditions

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.     

Growth,Feed Utilization,and Glycemic Response in European Sea Bass,Dicentrarchus labrax,Juveniles Fed Carbohydrate of Different Complexities

The effect of dietary carbohydrate complexity on growth, feed utilization, and glycemia was studied in European sea bass juveniles. Four isonitrogenous (50% crude protein) and isolipidic (15% crude lipids) diets were formulated to contain 20% pregelatinized maize starch (PGS diet), dextrin (DEX diet), maltose (MAL diet), or glucose (GLU diet). No effect of dietary carbohydrate complexity on growth was noticed. Feed efficiency and protein efficiency ratio were lower in fish fed the GLU diet than in the other groups, whereas the opposite was observed for feed intake. Plasma glucose peaked 3–4 h after feeding in fish fed the MAL and GLU diets, whereas in fish fed the PGS and DEX diets the peak was reached 5–6 h after feeding. Peak plasma glucose concentration (13 mmol/L) was higher in fish fed the GLU diet than the other diets (9 mmol/L). Shorter hyperglycemia duration was observed in fish fed the MALT and GLU diets (6 h) than the PGS and DEX diets (10 h). Complex carbohydrates delayed plasma glucose concentration peak compared with simple sugars, whereas the opposite was observed for hyperglycemia duration. Overall, dietary maltose, dextrin, and starch were apparently better utilized as energy source than glucose by European sea bass juveniles.     

Phosphorus requirement of European sea bass (Dicentrarchus labrax L.) juveniles

A growth trial was conducted to estimate the phosphorus requirement of European sea bass juveniles. Six experimental isonitrogenous and isoenergetic semi‐purified diets (casein based) were formulated to contain 0.48%, 0.65%, 0.77%, 0.86%, 1.05% and 1.25% phosphorus (diets D1, D2, D3, D4, D5 and D6 respectively). Dicalcium phosphate was used as dietary phosphorus source. Twelve groups of 25 fish of 10 g initial body weight were allocated to 55‐L tanks in a thermoregulated water recirculating system. Each experimental diet was assigned to duplicate groups of these fish. The trial lasted for 10 weeks and fish were fed two times a day, 6 days a week, to apparent visual satiation. At the end of the trial, final weight of fish fed diet D1 was significantly lower than that of the other groups, except of fish fed diet D4. Mortality of fish fed diet D1 was significantly higher than that of fish fed the other diets, except for diet D6. Feed efficiency and protein efficiency ratios were significantly lower with diet D1 than with the other diets. Nitrogen retention (% nitrogen intake) of fish fed diet D1 was significantly lower than in the other groups, except that of fish fed diet D4. Energy retention (% energy intake) was not significantly different among groups. At the end of the trial there were no differences in whole body composition among groups. Whole body phosphorus content averaged 0.72% (on a fresh weight basis) and was not significantly affected by dietary phosphorus content. Phosphorus retention averaged 6.1 g kg^?1 weight gain and was not significantly different among groups. Phosphorus retention (% phosphorus intake) was significantly higher in fish fed diets D2 and D3 than in fish fed higher dietary phosphorus levels. Expressed per unit body weight per day, phosphorus retention was not significantly different among groups fed diets D2 to D6, while phosphorus losses linearly increased with dietary phosphorus intake. Results of this trial indicate that the phosphorus requirement of sea bass juveniles was satisfied with a diet containing 0.65% of phosphorus.     

Assessment of rocky reef fish assemblages close to seaweed farming

The seaweed Kappaphycus alvarezii (Doty) Doty ex P. C. Silva, a red algal species, is the main global source of Kappa carrageenan. The introduction of such exotic species in regions outside their original locale can change the community structure of the areas into which they are introduced. The possible influence of seaweed farming on the rocky reef fish assemblage was assessed in Paraty, Brazil. The reef fish assemblage in the seaweed farming area was compared before and after the commencement of farming with two undisturbed control areas. Among the ten species ranked as the most frequently occurring in each area, eight were the same. The fish community structure close to the seaweed farming area did not change from that of the control areas, over the months of the study. Neither the fish diversity and richness indices, nor the total average abundance among areas was significantly altered. The average abundance of the different trophic groups varied over time in both the farm and control areas, revealing similar patterns. These results suggest that K. alvarezii can be cultivated up to at least 50 m from a rocky coast without altering the fish community structure of the surrounding waters.     

The effect of dietary protein replacement by crystalline amino acid on growth and nitrogen utilization of turbot Scophthalmus maximus juveniles

A study was conducted to evaluate the effect of partial replacement of dietary fish meal by crystalline amino acids on growth performance, feed utilization, body composition and nitrogen utilization of turbot juveniles.

Four diets were formulated to be isolipidic (12% DM) and isonitrogenous (8% DM). A fish meal based diet was used as control. In the experimental diets, a crystalline amino acid (AA) mixture was used to partially replace fish meal, corresponding to a non-protein nitrogen content of 19, 37 and 56%, respectively (diets 19AA, 37AA and 56AA, respectively). The overall amino acid profile of the experimental diets resembled that of the whole-body protein of turbot. Each experimental diet was fed to triplicate groups of 20 fish (initial body weight of 31.8 g) twice daily to apparent satiation for 42 days. During the trial water temperature averaged 18 °C.

Final body weight, weight gain (g kg ABW^− 1 day^− 1) and specific growth rate were not different between the control and 19AA diet but significantly decreased with the increase of crystalline-AA inclusion from 19 to 56%. Feed intake and feed efficiency of fish fed the control and diet 19AA were similar and significantly higher than those of fish fed the 56AA diet. At the end of the growth trial, there were no significant differences in whole-body composition among groups. Hepatosomatic index was also unaffected by dietary treatments.

Nitrogen retention (g kg ABW^− 1 day^− 1) of fish fed the control and the 19AA diets were similar and significantly higher than that of fish fed the other diets. Expressed as a percentage of the nitrogen intake, N retention was significantly higher with the control than with the 37AA and 56AA diets.

Daily ammonia excretion (mg kg ABW^− 1 day^− 1) of fish fed the control diet was significantly higher than that of fish fed the 37AA and 56AA diets, while daily urea excretion (mg kg ABW^− 1 day^− 1) did not significantly differ among treatments. Non-fecal nitrogen (ammonia + urea) excretion (mg kg ABW^− 1 day^− 1) was significantly higher for fish fed the control diet than in those fed the 37AA and 56AA diets. However, as percent of N intake, ammonia excretion and non-fecal N excretion were significantly higher in fish fed the 56AA diet than in those fed the control and 19AA diets.

Specific activity of glutamate dehydrogenase, alanine and aspartate aminotransferases did not significantly differ among experimental groups.

In conclusion, in diets with an overall amino acid profile resembling that of the whole-body protein of turbot, crystalline-AA may replace 19% of dietary protein without negatively affecting growth performances or feed utilization efficiency. However, higher protein replacement levels of protein-bound-AA by crystalline-AA severely depressed growth performance.     

Preliminary evaluation of pea seed meal in diets for gilthead sea bream (Sparus aurata) juveniles

Juvenile gilthead sea bream with a mean initial body weight of 5 g were fed for 12 weeks with experimental diets containing 10% and 20% fishmeal protein (sole protein source in the control diet) replaced by processed pea seed meals. The processed pea seed meals were dehulled, defibred, extruded and microground pea seed meal (PSM1) or whole pea treated by infrared radiation and ground (PSM2). Apparent digestibility coefficients of the experimental diets were determined in a separate trial. At the end of the growth trial there were no significant differences in growth performance, feed utilization or whole-body composition among experimental groups. There were no differences in apparent protein digestibility among experimental groups (except for fish fed PSM1 at the lowest inclusion level). Both dry matter and energy digestibility of the diets, including PSM2 and with the highest inclusion level of PSM1, were significantly lower than those of the control diet. The results of this study suggest that pea seed meal may replace up to 20% fishmeal protein in diets for gilthead sea bream juveniles without affecting fish performance. Further studies should focus on technological treatments to increase utilization of pea seed meal carbohydrate, as both apparent dry matter and energy digestibility were affected by dietary inclusion level and by pea seed meal processing method.     

Gut microbiota and gut morphology of gilthead sea bream (Sparus aurata) juveniles are not affected by chromic oxide as digestibility marker

The present work aimed to evaluate whether the use of chromic oxide (Cr₂O₃) as dietary inert marker in fish digestibility studies interferes with gut microbial community modulation and gut morphology. To assess the effects of Cr₂O₃ under potential diverse microbiota populations, dietary Cr₂O₃ was tested using challenging plant feedstuffs (PF)‐based diets supplemented or not with prebiotics, as prebiotics are expected to modify gut microbiota populations. For that purpose, three diets were formulated to include circa 20:80 fish meal and PF as protein sources, without (CTR) or with prebiotic supplementation (10 g/kg XOS or GOS). These diets did not include Cr₂O₃ (?Cr₂O₃ diets). Three similar additional diets were formulated to include 5 g/kg Cr₂O₃ (+Cr₂O₃ diets). Cr₂O₃ effects on gut microbiota were assessed for the first time in the allochthonous (digesta) and autochthonous (mucosa) community by a culture‐independent molecular approach, using denaturing gradient gel electrophoresis (DGGE). No differences in gut bacterial profiles (number of operational taxonomic units, microbiota richness, diversity and similarity indices) were observed between dietary treatments. No significant alterations in submucosa layer structure, enterocytes and eosinophilic granular cells structure, goblet cells and leucocytes quantity were detected in the distal intestine among diets. In conclusion, data indicate that dietary inclusion of 5 g/kg Cr₂O₃ does not interfere with gilthead sea bream (Sparus aurata) gut microbiota and gut morphology, suggesting that a dietary incorporation level of 5 g/kg Cr₂O₃ can safely be used as inert marker in digestibility studies.