Amod Kulkarni

A Microbial Feed Additive Abates Intestinal Inflammation in Atlantic Salmon

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.

Detection of Francisella piscicida in Atlantic cod (Gadus morhua L) by the loop-mediated isothermal amplification (LAMP) reaction

The loop-mediated isothermal amplification (LAMP) reaction was evaluated for its speed and sensitivity in detecting the presence of Francisella piscicida, the causative agent of francisellosis in Atlantic cod (Gadus morhua). Four primer sets, consisting of two outer and two inner, were designed from the groEL gene of the pathogen. The LAMP reaction was optimised at 63 degrees C for 1h using bacterial genomic DNA as the template and the products were visualised under ultra-violet light and analysed by agarose gel electrophoresis. A ladder-like pattern of bands, specific for F. piscicida, was amplified from positive samples. The method was highly specific for the detection of F. piscicida and was 100 times more sensitive than conventional PCR. In addition, the LAMP assay was able to detect the pathogen in kidney and splenic samples of naturally-infected Atlantic cod.

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: an uptake and processing study in the midgut of Penaeus monodon.

Several 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.

Recognition of purified beta 1,3/1,6 glucan and molecular signalling in the intestine of 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 metabolism-related) 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.

Expression profile of bio-defense genes in Penaeus monodon gills in response to formalin inactivated white spot syndrome virus vaccine

White spot syndrome virus (WSSV) is the most devastating pathogen of penaeid shrimp. While developing technology to vaccinate shrimp against WSSV, it is imperative to look into the immune response of the animal at molecular level. However, very little information has been generated in this direction. The present study is an attempt to understand the expression of bio-defense genes in gill tissues of Penaeus monodon in response to formalin inactivated WSSV. A WSSV vaccine with a viral titer of 1×10(9) DNA copies was prepared and orally administered to P. monodon at a rate of 1.75×10(6) DNA copies of inactivated virus preparation (IVP) day(-1) for 7days. The animals were challenged with WSSV on 1st and 5th day post vaccination, and temporal expression of bio-defense genes in gill tissues was studied. Survival of 100% and 50% were observed respectively on 1st and 5th day post vaccination challenge. The humoral immune genes prophenoloxidase (proPO), alpha 2-macroglobulin (α2M), crustin and PmRACK, and the cell mediated immune genes caspase and Rab7 were up regulated in gill tissue upon vaccination and challenge. The expression of humoral gene crustin and cellular gene Rab7 was related to survival in IVP administered shrimp. Results of the study suggest that these genes have roles in protecting shrimp from WSSV on vaccination.