Pathophysiological changes associated with dietary melamine and cyanuric acid toxicity in red tilapia

This study examined the adverse effects of feed-delivered melamine (MEL) and cyanuric acid (CYA) in red tilapia. Diet 1 (without MEL and CYA), diets 2–4 (with MEL and CYA at 2.5, 5 and 7.5 g kg⁻¹ diet, respectively) and diets 5 and 6 (with either MEL or CYA at 10 g kg⁻¹ diet) were examined. MEL alone lowered both growth and FCR (P < 0.05), and CYA alone reduced the FCR of tilapia. Protein efficiency ratio and apparent net utilization of fish on diets 2–6 were poor (P < 0.05). The renal tubules of fish ingested MEL-CYA combination had melamine–cyanurate crystals. On the other hand, diets with only one chemical did not induce such crystals. MEL and CYA in whole body, fillet or viscera reflected their dietary inclusion levels. The levels of Hsp70 were increased in the liver of fish that ingested MEL and CYA, in combination or singly (P < 0.05). However, in the kidney, such an increase was visible only in the fish that received diet 4 (P < 0.05). Combination of MEL and CYA at inclusion levels > 5 g kg⁻¹ diet induced the activity of catalase in liver and the activity of glutathione peroxidase in liver and kidneys. Therefore, these adulterants should not be included in fish feeds.     

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.     

Antagonistic activity of bacterial isolates from intestinal microbiota of Atlantic cod, Gadus morhua, and an investigation of their immunomodulatory capabilities

In an effort to identify potential probionts, four bacterial strains obtained from the intestinal tract of wild-caught Atlantic cod, Gadus morhua , were tested for their antagonistic activity against Vibrio anguillarum and Aeromonas salmonicida , two of the most common bacterial pathogens in cod aquaculture, using a well-diffusion agar assay at two incubation temperatures: 13 and 20 °C. The tested bacteria exhibited dissimilarity in their inhibitory action against the target pathogens – an enhanced activity was particularly observed for strains GP11 and GS11 at the highest incubation temperature. Based on the 16S ribosomal DNA gene sequence analysis, the strains showed high similarity to Psychrobacter sp. (strain GP11), Shewanella sp. (GS11), Photobacterium sp. (GP31) and Vibrio sp. (GV11). The incubation of Atlantic cod head kidney cells with the heat-inactivated probiotic strains resulted in the differential expression of immune-response genes that are related to bacterial defence and inflammation. Thus, the gut-derived bacterial strains have probiotic potential and their possible immunomodulatory capabilities could be determined under in vitro conditions.     

Simultaneous detection of pathogens causing francisellosis,furunculosis and vibriosis in Atlantic cod by multiplex polymerase chain reaction

An assay for the simultaneous detection of three major bacterial pathogens of Atlantic cod with multiplex polymerase chain reaction (mPCR) was developed. Primers targeting the flanking regions of genes groEL, gyrB and amiB were standardized to diagnose francisellosis, furunculosis and vibriosis respectively. The detection limit of Francisella piscicida, Aeromonas salmonicida and Vibrio anguillarum in the mPCR assay ranged from 10 ng to 100 pg of bacterial DNA mL^?1, 10 μg to 50 ng of bacterial DNA mL^?1 and 1 μg to 100 ng of bacterial DNA mL^?1 respectively. These primers were found to be specific in the determination of the respective genes in the individual pathogen without cross‐reaction, thus making the assay an efficient tool to detect the presence of these pathogens simultaneously or individually.     

Real‐time quantification of the immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus

Whether it is better to use viable or non‐viable probionts in aquaculture is still a matter of debate. In this study, the molecular immunomodulation in rainbow trout Oncorhynchus mykiss induced by viable or killed forms of the probiont Lactobacillus rhamnosus JCM 1136 was investigated. Three forms of this probiont: (1) heat‐killed (HK), (2) live spray (LI) and (3) freeze‐dried (FD) were incorporated into a basal (control) diet for rainbow trout O. mykiss. The LI and FD diets are referred to as viable diets. A rearing trial, in triplicate, was conducted for 30 days, with the control and probiotic diets as treatments. The cytokine genes such as the tumour necrosis factor (TNF), transforming growth factor (TGF‐β), interferon (IFN) and immune gene Immunoglobulin (Ig) found in tissues from the kidney and spleen were assessed for their expression pattern by real‐time polymerase chain reaction. The tested immune genes were up‐regulated in the treatment groups, sometimes even in many folds like in the case of the Ig gene. The TNF gene was found to be highly (P<0.05) up‐regulated (5000‐fold) in groups fed both viable forms (LI, FD). With regard to the TGF‐β gene, the spleen of the HK and FD groups showed significant up‐regulation of 20‐ and 30‐folds respectively. The IFN gene was up‐regulated (P<0.05) in all treatments, but more in the viable diet treatments. Kidney and spleen tissues showed similar expression patterns, i.e. all of these genes were up‐regulated more with the viable diets that with the control, and in most cases, the viable diets induced a higher expression of the immune genes than the HK diet.     

Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136

This study was undertaken to examine the effect of supplementing a suggested probiotic bacteria Lactobacillus rhamnosus JCM 1136 in feed on immune response and gut flora composition of rainbow trout Oncorhynchus mykiss. The probiotic bacteria were incorporated into a commercial feed to constitute two experimental diets containing either 10(9) or 10(11) colony forming unit of live bacteria/g of feed while a third diet without the bacterial supplement served as the control diet. The diets were offered to rainbow trout (75g average weight) in triplicate tanks for 30 days. Fish were sampled at 10, 20 and 30 days after commencement of the feeding trial to determine the proportion of the given probiont in the gut microflora composition and the nonspecific humoral and cellular immune responses on the 30th day. The relative proportion of the probiont increased with the feeding duration in the intestine, but not in the stomach. The proportion of L. rhamnosus in the stomach corresponded to the intake levels while no such relation existed in the intestine. The serum lysozyme and complement activities were significantly greater in fish fed the higher level of probiont compared with the control fish. The phagocytic activity of head kidney leucocytes also showed similar tendencies. These observations indicate the potential immuno-regulatory role of probiotic organisms in rainbow trout.