Location: Warmwater Aquaculture Research Unit2011 Annual Report
1a. Objectives (from AD-416)
Research will address methods to determine the presence of pathogens in catfish/catfish products and to maximize elimination methods. Detection techniques will be developed to aid in processing and packaging operations, which should further enhance product safety. Specifically the new objectives are: 1)Optimize safety of aquaculture products through innovative processes for reducing microbiological, physical and chemical hazards in seafood/aquaculture products. 2)Determine the mechanisms influencing microbial survival of selected pathogens in seafood/aquaculture products. 3)Optimize the market value of seafood/aquaculture products through enhanced food safety and quality.
1b. Approach (from AD-416)
These funds will be used for 1) Extramural research with Mississippi Center for Food Safety and 2) Aflatoxin research.
3. Progress Report
Research related to control of pathogens in food products, in particular Listeria monocytogenes, is focused on developing strategies for reducing pathogens in catfish, seafood, and other meat products. New generally regarded as safe (GRAS) antimicrobials for control of bacterial pathogens in aquaculture products are being developed and tested for their effectiveness. Effects of irradiation on shelf-life extension and pathogen reduction are being determined for several foods, including catfish. A rapid, inexpensive antibody-based assay is being developed for detecting botulism neurotoxin in catfish. This assay will be useful for screening purposes. A luciferase reporter gene has been inserted into Salmonella enterica and selectively produces light in viable cells. This technique allows real-time detection of the bacteria at low levels and allows monitoring of bacterial progression under different environmental and chemical conditions. Training of catfish producers and processors was conducted to reduce potential biosecurity and food safety risks. These research projects will play a role in assuring safe, high-quality food products to consumers and will be important to maintaining viable aquaculture and seafood industries in the U.S.
1. Inactivation of Salmonella enetrica on cantaloupes and mangos by X-ray. Fruits including cantaloupe and mango are an essential part of healthy eating. The United States Department of Agriculture recommends that consumers include more fruit and vegetables in their diet to decrease the risk of cardiovascular disease and cancer. As produce consumption has increased, a significant increase in the number of foodborne disease outbreaks and illnesses associated with fresh produce has been reported. Many outbreaks, especially those caused by the bacterial pathogen Salmonella, have been associated with cantaloupe and mango. Mississippi State University scientists determined effects of X-ray on reduction of bacteria and shelf-life in cantaloupes and mangos inoculated with Salmonella. Salmonella was reduced more than 10,000,000 cells per gram of cantaloupe and mango with 2.0 and 1.0 kGy X-ray doses. In addition the inherent microflora (other bacteria, yeasts, and molds) was reduced to less than 10 cells per gram. This research demonstrates X-ray irradiation is an effective method for reducing pathogens and microflora in cantaloupes and mangos and has important implication for food safety.
2. Inactivation of Salmonella, Escherichia (E.) coli O157: H7 and Shigella species on parsley and green peppers by X-ray. Several recent foodborne disease outbreaks associated with vegetables including parsley and peppers have been reported. Outbreaks have been associated with bacterial pathogens Salmonella, Escherichia coli O157:H7 and Shigella spp. Mississippi State University scientists studied effects of X-ray doses (0.1, 0.5, 1.0 and 1.5 kGy) on parsley and green peppers inoculated with Escherichia coli O157:H7, Salmonella enterica and Shigella flexneri. The effect of X-ray on microflora counts (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated parsley and green peppers was also evaluated. Pathogenic bacteria were nearly emlinated on green peppers and parsley with 1.0 and 1.5 kGy X-ray. Furthermore, treatment with X-ray significantly reduced the initial inherent microflora on parsley and green peppers and throughout refrigerated storage for 30 days. This research demonstrates significant benefit to consumers through use of X-ray to reduce pathogens and extend shelf-life in parsley and green peppers.
3. Reduction of Listeria monocytogenes on smoked salmon and catfish using X-ray treatment. Listeria monocytogenes is a foodborne pathogen that causes severe disease in humans. Listeria monocytogenes is widely found in the environment and has been isolated from seafood products. L. monocytogenes can easily be destroyed by cooking in contaminated seafood; however its presence in ready-to-eat seafood, particularly smoked fish, is a safety concern. Mississippi State University scientists determined the efficacy of X-ray irradiation on the inactivation of Listeria monocytogenes and shelf-life of smoked salmon and catfish. Smoked catfish and salmon fillets were inoculated with a 3-strain mixture of Listeria monocytogenes and treated with 0.1, 0.5, 1.0 and 2.0 kGy X-ray. Un-irradiated and irradiated samples were stored at 5°C for 35 days and periodically tested for L. monocytogenes counts. X-ray of 1.0 kGy significantly reduced all bacteria, including L. monocytogenes. X-ray of 2.0 kGy extended shelf-life to 35 days in smoked salmon and 30 days in smoked catfish. This research demonstrated X-ray irradiation can be used to improved safety and extended shelf-life of smoked fish and is beneficial to consumers.
4. Characterization of Campylobacter and E. coli in a real-time bioluminescent assay. Catfish, red meat and poultry processing fall under the authority of the United States Department of Agriculture Food Safety Inspection Service. The goal of Mississippi State University scientists is to assist the food animal industry in meeting current and future federal safety regulations. As part of this effort we have established and tested an in vitro real-time bioluminescent-based reporter system for monitoring Salmonella and Escherichia coli in food production and processing environments. This system is based on the light-producing gene (lux) which is inserted into the bacterial chromosome causing emission of light and allows rapid, efficient detection of these pathogens. We demonstrated this system was effective for tracking Salmonella and E. coli on refrigerated raw catfish fillets, boneless chicken fillets, and ground beefs. Using the reporter system, we evaluated three antimicrobial compounds for their efficacy to inhibit growth of E. coli. We also initiated work to allow us to study the attachment of Salmonella to chicken skin. Our work demonstrates the utility of this reporter system for use in studying these foodborne pathogens. Understanding this mechanism will aid in characterizing persistence of Salmonella and E. coli, two common and important food pathogens, in production and processing environments.
5. Prevention of potential food quality problems associated with an emerging larval nematode in commercial catfish meat. Recently unidentified larval nematodes were found in the skin and muscle of commercial catfish, causing lesions that compromised the quality and appearance of the whole meat and fillet product. These parasites were tentatively identified as drucunculoids with aquatic turtles, fish and aquatic invertebrates as hosts in the life cycle of these parasites. The objectives of this project were to: confirm the host (turtle species) involved in the life cycle of this unknown larval nematode; identify the invertebrates that serve as intermediate hosts for the larval stages of this parasite, and develop polymerase chain reaction (PCR)-specific probes to detect the life stages of this parasite in the invertebrate hosts. The parasite was sequenced and found to be 98% homologous with the spiruroid Camillanus (C.) oxycephalus confirming that red-eared sliders in catfish ponds are infected with at least one nematode, S. trispinosum, which has the potential to infect catfish. Invertebrate collections were also done in additional ponds (n=5) with histories of poor performance, and no larval nematode stages were found in these ponds. We determined that red-ear slider turtles are infected with a nematode that also infects fish. We have now generated sequencing data both on this parasite and the unknown drucunculoid-like nematode which can be used in the development of PCR primers that will be more efficient and sensitive in the detection of other stages of these parasites in the fish and invertebrate hosts. This will provide the diagnostic tools necessary for the development of management programs to control/eradicate these invertebrates and interrupt the life cycle prior to infection, preventing the encystment of these larval stages in the catfish tissue.
6. Assessment, development and implementation of training materials for food defense/safety and biosecurity issues within the catfish processing industry. Agriculture has been listed as a potential target for terrorism in which the security of our food supply may be breached. Food defense and traceability plans are designed to evaluate all areas of a business and infrastructure including personnel, ingredients, supplies, transportation, processing, distribution, and product tracking. The objective of this research and subsequent training sessions was to promote awareness and educate catfish farmers and processors on the principles of food defense, biosecurity and traceability of agricultural commodities and ingredients used in human food production. Training sessions were provided at a choice of two different dates and locations. Participants were asked a series of questions involving different areas of food defense, safety, biosecurity, and traceability. Of those surveyed, 74% said they have a food defense plan; 48% had a working traceability plan; and 52% had a biosecurity plan for catfish. Additionally, a pre-and post-assessment were given to evaluate the effectiveness of the training sessions. Overall 68% of participants earned an equal or higher score on the post-assessment. Training will continue through seminars, workshops, supplementary materials, and checklists. While many larger and corporate structured enterprises in aquaculture were found to be more prepared on certain principles, there is a need to advance training and knowledge. With proper training and awareness, farmers and others throughout the chain are better prepared to continue, and even increase, the security of our food supply.
7. Use of acid and sweet whey as edible coating to control oxidative deterioration of catfish fillets. Retail value of U.S. farm grown catfish was estimated to be over $1.56 billion in 2005 and is an important contributor to the economies of numerous southern states including Mississippi. Unfortunately, oxidative degradation resulting in off-color and flavor development starts once any muscle foods are processed. Hence, there is significant commercial interest in identifying food-grade antioxidants for use as coating agents. We determined that acid (cottage cheese) and sweet (Edam and Cheddar) whey could effectively and inexpensively be used in edible coatings to control the oxidative deterioration of catfish fillet. In addition we found that controlled heat treatment (thermization) enhanced antioxidative capacity of whey based on measurement of protein degradation indicators in catfish fillets. Over a storage period of 14 days (2°C), all treatments dramatically and favorably affected these indicators (e.g., average 66% reduction by the treatments in amines compared to uncoated control). Colorimetry indicated significant improvement in fillet color in all treatments with Cheddar whey being the best. Moisture loss during storage was reduced an average of approximately 30% in all treatments with Cheddar whey again being the best. This was speculated to be due to greater amount of casein peptides and free amino acids in Cheddar whey. Data indicate that natural, nutritious, inexpensive and abundantly available whey, with or without thermization, can significantly enhance shelf quality and benefit the catfish industry and consumers.
8. The development of an ultra-sensitive analytical method to detect Botulinum toxin contamination of catfish. Visceral toxicosis of catfish (VTC) is a relatively new disease syndrome that affects food size catfish raised in commercial catfish ponds. Over the past ten years losses associated with this disease have been estimated to be in excess of one million pounds. Serum neutralization studies and endopep mass spectrometry indicate the disease is a form of botulism, caused by a toxin produced by the bacterium Clostridium botulium. Development of a sensitive, low cost, toxin free assay for measuring catfish toxin-specific antibody production will be useful for monitoring and managing this disease. We continued work to optimize extraction of Botulinum neurotoxin (BoNT) from catfish serum, muscle and intestinal tract; improve sensitivity and quantitative detection capabilities for BoNT; and initiated development of methods to study intact BoNT. Fish muscle/solvent ratios for BoNT extraction were optimized and the serum protein sample preparation was modified by filtration which increased BoNT detection via liquid chromatography-mass spectrometry/mass spectometry (LC-MS/MS). Application of new technology and software allowed detection of the toxin at the 166 pg level. Work with BoNT is complicated by the toxicity of the intact protein. The model protein Proteinase K was selected and is being used to test technologies and develop protocols that can be used to study intact BoNT. This research will provide insights into monitoring and managing VTC in catfish.
9. New generally regarded as safe bacteriophage LISTEXTM P100 for reduction of Listeria monocytogenes in aquaculture products. Listeria monocytogenes is a deadly foodborne pathogen. Freshwater and marine fish are sometimes inadvertently contaminated with very low levels of L. monocytogenes. Intervention/elimination strategies for L. monocytogenes in processed, raw and smoked seafood products are currently inadequate. Recently the Food and Drug Administration and the United States Department of Agriculture approved a novel antimicrobial, bacteriophage Listex P100, which is commercially available and has generally regarded as safe (GRAS) status for the application on both raw and ready-to-eat food products to control L. monocytogenes. Mississippi State University scientists’ research results reveal a strong bactericidal activity of bacteriophage P100 phage against L. monocytogenes on raw salmon fillets. Previous studies demonstrate that the L. monocytogenes cells present in a biofilm matrix show greater resistance to antimicrobial agents than those present in planktonic conditions. Our work demonstrated the potential of phage treatment as an alternative strategy for the control of L. monocytogenes biofilms on the stainless steel surfaces. This technology has potential to improve control of L. monocytogenes in seafood processing environments.
10. Identification and evaluation of attachment of Listeria monocytogenes on the surface of ready-to-eat products. Listeria (L.) monocytogenes is well known to possess a strong attachment and biofilm formation abilities. The bacterium has unique ability to attach to various ready-to-eat (RTE) surfaces and processing equipment which makes it difficult to prevent the cross contamination during food processing. Mississippi State University scientists determined surface proteins and virulence factors of L. monocytogenes associated with attachment on vegetables. Activated listerial cells were inoculated on lettuce leaves and incubated at 4°C. We tested 31 genes encoding surface proteins and 8 genes encoding virulence factors of L. monocytogenes using real-time PCR. The transcriptional levels of some surface proteins on the lettuce leaf were up-regulated after inoculation. However, the gene expression of virulence factors did not change. The results suggest that cell surface proteins and sigB (a key regulator of general stress) may be involved in attachment of Listeria to surfaces. This study will help us understand how L. monocytogenes survive or grow on the surface of leafy vegetables. The information will help preventive strategies to reduce the contamination of L. monocytogenes on vegetables.Wang, C., Chou, C., Tseng, C., Ge, X., Pinchuk, L.M. 2011. Early gene response of human brain endothelial cells to Listeria monocytogenes. Canadian Journal of Microbiology. 57:441-446.