The efficacy of a microbial feed additive (Bactocell®) in countering intestinal inflammation in Atlantic salmon was examined in this study. Fish were fed either the additive-coated feed (probiotic) or feed without it (control). After an initial 3-week feeding, an inflammatory condition was induced by anally intubating all the fish with oxazolone. The fish were offered the feeds for 3 more weeks. Distal intestine from the groups was obtained at 4 h, 24 h, and 3 weeks, after oxazolone treatment. Inflammatory responses were prominent in both groups at 24 h, documented by changes in intestinal micromorphology, expression of inflammation-related genes, and intestinal proteome. The control group was characterized by edema, widening of intestinal villi and lamina propria, infiltration of granulocytes and lymphocytes, and higher expression of genes related to inflammatory responses, mul1b, il1b, tnfa, ifng, compared to the probiotic group or other time points of the control group. Further, the protein expression in the probiotic group at 24 h after inducing inflammation revealed five differentially regulated proteins – Calr, Psma5, Trp1, Ctsb, and Naga. At 3 weeks after intubation, the inflammatory responses subsided in the probiotic group. The findings provide evidence that the microbial additive contributes to intestinal homeostasis in Atlantic salmon.

Atlantic salmon was orally intubated with a highly purified β-glucan product (MacroGard®) to study the recognition of the molecule by the receptor genes, the regulation of the downstream signalling genes and global proteins, and the micromorphological changes in the intestine. The β-glucan receptor genes of Atlantic salmon, sclra, sclrb, sclrc and cr3, seem to recognize the molecule, and initiate the downstream ITAM-motif signalling, as evident from the significantly high mRNA levels of ksyk, mapkin2, il1b and mip2a levels. Among the altered proteins, the Apoa4 (involved in carbohydrate and lipid metabolism); Tagln, Actb (uptake of β-glucan); Psma2 (associated with substrate recognition); and Ckt (energy metabolismrelated) were the overexpressed ones. The underexpressed proteins included the Uk114, Rpl9, Ctsb and Lgal that are connected to proliferation, LPS-stimulation, Il1b and lactose recognition, respectively. Furthermore, the mRNA levels of igt and the number of immune cells in the distal intestine were found to increase upon β-glucan uptake by the fish. This study provides some clues on the mechanisms by which the β-glucan evokes response in Atlantic salmon, particularly at the intestinal level.

Crustacean aquaculture, dominated by shrimp, is a highly profitable food-producing sector in the world. However, a variety of biotic and abiotic stressors can have adverse effect on the immune system of shrimp making them susceptible to diseases. Although a vertebrate-like adaptive immune system is lacking in shrimp, an efficient innate immune system renders protection against invading pathogens. The innate immune system comprises two distinct but overlapping components, the cellular and humoral, and these are regulated through several signal transduction pathways. The signal pathways are initiated by the recognition of pathogen-associated molecular patterns by germline-encoded pattern recognition receptors leading to the production of different effector molecules that act against the pathogens. RNAi-mediated post-transcriptional gene silencing and microRNA regulation of immune response have also been found to be functional in shrimp. Similarly, apoptosis and apoptosis-related genes are also reported, besides interferon (IFN) system-like antiviral regulatory mechanism. Further, some form of immune memory, termed 'immune priming' or 'innate immunity with specificity' and 'quasi-immune response' is recorded in shrimp and these abilities have been exploited in verifying the immunoprotection against different pathogens. Antigens developed either directly from the pathogens or through recombinant proteins have been tested for immune-protective ability. RNAi-mediated protection has also been demonstrated against different shrimp viruses. This review summarizes the available scientific information on immune responses and the immunoprotection trials carried out in crustaceans with a focus on shrimp. The available research evidences indicate the potential of developing effective immunoprophylactic measures in shrimp.

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

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A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL(-1) crude WSSV protein, respectively. The FTA could be completed in 8-10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4-8 °C. The FTA is available as a rapid test kit called 'RapiDot' for the early detection of WSSV under field conditions.

a b s t r a c tSeveral oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.

A loop‐mediated isothermal amplification (LAMP)‐based assay was developed for the detection of atypical furunculosis, caused by Aeromonas salmonicida in Atlantic cod, Gadus morhua. Gene gyrB encoding the B subunit of DNA gyrase present in the pathogen was selected for designing five sets of primers targeting the flanking regions of the gene. The primers were specific for the detection of A. salmonicida with no cross reactions to other bacterial pathogens commonly infecting Atlantic cod, e.g., Vibrio anguillarum, Francisella piscicida, Yersinia ruckeri and some endogenous bacteria found in the gut of Atlantic cod. The detection limit of the assay was 1 picogram of bacterial DNA mL−1, whereas there was a decrease in detection limit by 1 log dilution in the presence of mucus as inhibitor. Because of its specificity and sensitivity, LAMP can be considered a useful tool in routine surveillance programs in aquaculture systems for monitoring atypical furunculosis in Atlantic cod and other marine species. PRACTICAL APPLICATIONS LAMP is a potential diagnostic technique that can be used for the rapid and early detection of atypical Aeromonas salmonicida, the causative agent of furunculosis in Atlantic cod. This technique does not require sophisticated equipment and can be performed under isothermal conditions. The assay is highly sensitive to enable detection of the pathogen prior to the onset of infection, thus, mitigating measures can be applied to prevent heavy losses of the cultured stock.