Location: Warmwater Aquaculture Research Unit2013 Annual Report
1a. Objectives (from AD-416):
1) Develop and/or improve novel methods for the detection, identification and quantification of pathogens and chemical residues in catfish and other products; 2) Quantification of high-risk and low-risk Listeria monocytogenes serotypes and other pathogens on catfish environment and products by molecular tools such as multiplex Polymerase Chain Reaction (PCR) and surfaced enhanced Raman microscopy; 3) To develop novel, sustainable processes and products from catfish and its sub-products so as to enhance efficiencies while diminishing environmental impact; 4) To develop and apply novel bioactive compounds from catfish sub-products and to apply these and other compounds to improve heatlh and other functionalities (antimicrobial, antioxidant); and 5) To generate sound risk assessment data for establishing/modifying food safety regulations and to reduce/eliminate food safety problems in the aquaculture industry and their environemnt.
1b. Approach (from AD-416):
Detection and quantification of pathogens such as salmonella and Listeria and others is of priority as per literature and preliminary risk assesments conducted on catfish and aquaculture products. Novel detection and quantification methods with rapid and economical applicability to the industry are to be developed and tested in the field. These methods will be targeted as easy to be done, require minimum expertise and facilities and be rapid and accurate. In addition testing methods for therapeutic drugs are needed to test domestic and imported products reaching consumers. These tests will also be rapid and accurate. The discovery of genetic traits of Listeria monocytogenes in catfish and related aquaculture products will help in identification of the most proliferant pathogen in catfish and may help in identifying possible resistance mechanisms. Sound risk assesments need to be developed to prioritize the risks of domestic and imported catfish and aquaculture products. Databases will be complemented to develop risk assesments and risk management plans to minimize food safety and health concerns with catfish and aquaculture products. Novel products and sub products are needed to enhance the health benefits of catfish, diversify market available items, utilize more than 60% of the product that is underutilized now, and enhance product shelf life and safety. Native and natural compounds will be identified and applied pre- and post-harvest to study functionality and possible health benefits of catfish products for humans and pets. In addition, these processes and products will be developed to favor a sustainable chain in pre- and post-harvest life of catfish.
3. Progress Report:
The results indicated that the 0.1 and 0.2 kGy X-ray treatments significantly reduced the Salmonella populations by 2.9 and 3.2 log reduction on tuna samples. Biomarkers on Vibrio vulnificus and Salmonella were discovered and optimized on its expression in 24 hours. Research Collaboration Agreement (RCA) between the Food and Drug Administration (FDA) of the United States Government and Mississippi State University (MSU) is being developed. MSU Scientists have been successful in developing polymerase chain reaction (PCR) primers that can differentiate serotypes 4d and 4e from serotype 4b. Four primer sets were tested, and one set was consistent (ATCC19117_2265). Previously, MSU Scientists had identified primers based on Lmof2365_2059 that separates serotype 1/2b from the other serotypes. Based on Mauve alignment, we determined that the genome sequences of serotypes 1/2c and 3c are highly identical and design of PCR primers to differentiate them will be challenging. In 2013, several primer sets were tested to differentiate serotypes 1/2a and 3b. None have been consistent. Eight new primer sets are currently being tested. Identification and evaluation of attachment of Listeria monocytogenes on the surface of ready-to-eat products have been started. MSU Scientists have screened 32 genes encoding surface proteins and lipases of the strain to find highly expressed genes on lettuce leaves. Results showed that transcription levels of five genes were significantly up-regulated on lettuce leaves. In silico analysis showed that LMOf2365_0859 contains a putative cellulose binding domain. Thus, we hypothesized that this gene may be involved in an attachment to vegetables and named Listeria cellulose-binding protein (LCP). MSU generated a LCP mutant (LCP) by deleting the LCP gene. The LCP lost its ability to attach on the surfaces of leafy vegetables and cantaloupe skin. Fluorescence microscopy and field emission scanning microscopy analysis further support these findings. Results strongly suggest that LCP plays an important role in an attachment to vegetables and fruits. Persistence of Salmonella biofilms within food processing environments is an important source of Salmonella contamination in the food chain. In this study, essential oils of thyme and oregano and their antimicrobial phenolic constituent carvacrol were evaluated for their ability to inhibit biofilm formation and inactivate preformed Salmonella biofilms. Of the three Salmonella strains, Salmonella Typhimurium ATCC 23564 and Salmonella Typhimurium ATCC 19585 produced stronger biofilms than Salmonella Typhimurium ATCC 14028. Biofilm formation by different Salmonella strains was 1.5- to 2-fold higher at 22°C than at 30 or 37°C. The presence of nonbiocidal concentrations of thyme oil, oregano oil, and phenolic carvacrol at 0.006 to 0.012% suppressed Salmonella spp. biofilm formation 2- to 4-fold, but could not completely eliminate biofilm formation. Reduction of biofilm cell mass was dependent on antimicrobial concentration. A minimum concentration of 0.05 to 0.1% of these antimicrobial agents was needed to reduce a 7-log Colony forming units biofilm mass to a nondetectable level on both polystyrene and stainless steel surfaces within 1 h of exposure time. MSU Scientists are using novel proteomic techniques to discover new biomarkers in chicken and catfish relevant in foodborne disease, such as chicken salmonellosis. MSU Scientists are also discovering new active enzymes (kinases and deubiquitinases), which are important in chicken and catfish, but the developed techniques will also be relevant for agricultural research on other animals. MSU Scientist have developed and optimized an animal tissue processing method, which is compatible with the chemical proteomics to uncover novel, previously unidentified kinases and deubiquitinases, and quantitation thereof in infection process. MSU Scientists have used Covaris, which is an instrument based on focused ultrasounds, to disturb tissue and lyse the cells, at the same time retaining the catalytic activity of enzymes relevant to our study. MSU Scientists were able to use mass spectrometry to identify some of the novel enzymes in animals in tissue and in in vitro-cultured cells, and are currently cloning three of these genes, which showed regulation during Salmonella infection, into an expression vector to develop future studies on these genes. Further, measured inhibition of animal UCHL5 in HD11 macrophage cells, and are currently cloning the UCHL5 gene into an avian expression vector to further uncover its function. MUS Scientists also showed that by knocking down expression of UCHL5 we can manipulate the number of bacteria surviving within the macrophage, and that by using a UCHL5-selective inhibitor (small molecule-based) MSU Scientists can achieve a similar effect. MSU Scientists also showed that this effect is not due to proteasomal inhibition. Overall, were able to fulfill most of the planned work for this budgetary period. The quality attributes, descriptive sensory characteristics, and muscle proteomes (sarcoplasmic and myofibrillar) of normal and reddish channel catfish (Ictalurus punctatus) fillets were evaluated. Reddish fillets had greater redness and yellowness values and less lightness than normal fillets, but no differences existed in cooking loss and instrumental texture. The reddish fillets were more bitter, earthy, grassy, metallic, and oxidized, and had more off-flavor than normal fillets. Profiling of muscle proteomes employing 2-DE and MS identified several myofibrillar and sarcoplasmic proteins. Analyses of the sarcoplasmic proteome revealed differential abundance of hemoglobin (beta), which was over-abundant in reddish fillets. Previous studies have not primarily focused on understanding the protein expression in catfish and its effect on final catfish quality. Therefore, in the present study, we identified the protein biomarkers responsible for redness in catfish fillets. Further research will focus on understanding catfish protein expression with emphasis on pre- and peri-mortem factors to determine the influence of the pre-harvest environment on fillet quality.