Location: Warmwater Aquaculture Research Unit2007 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. Original start and term dates 5/14/93-5/13/98, then 05/14/98 - 05/13/03, then 05/14/03 - 05/13/08.
3. Progress Report
Effects of freezing and duration of refrigeration prior to freezing on levels of Vibrio vulnificus in oyster meat. Pathogenic strains of Vibrio vulnificus are natural inhabitants of estuarine environments world wide and can be transmitted to humans through consumption of raw shellfish which flourish in the same estuaries. Until recently, most post harvest processes for preserving and extending shelf-life of in-shell and shucked oysters have centered on refrigeration, but research to find other processes to eliminate this pathogen has expanded. Recent information gained through validation studies of freezing (individually quick frozen (IQF)) (Schwarz, 2003) has indicated variability in success of freezing depending on storage conditions prior to freezing. Cryogenic freezing successfully reduced Vibrio vulnificus numbers, with an adjusted gross mean (AGM) of 87,000 MPN/g oyster meat, to < 30 MPN/g oyster meat in 7 days and to non-detectable levels (<3 MPN/g oyster meat) in 35 days. However, there was no difference in reduction of Vibrio vulnificus in oysters refrigerated for different times up to 4 days prior to freezing, indicating Vibrio vulnificus did not become more cold tolerant or more process resistant after refrigeration. Knowledge gained from this study will provide specific information for industry partners interested in processing using IQF freezing technology. Establishing specific guidelines for transport and storage of oysters deemed for IQF processing will maximize the effect of freezing on vibrio reduction in half shell oysters. This research directly addresses National Program 108 – Food Safety, Component 1.2 – Pathogens, Toxins and Chemical Contaminansts – Postharvest; Problem Statements 1.2.4 Processing Intervention Strategies and 1.2.6 Safety and Health. Genome sequencing of L. monocytogenes serotype 4a strain HCC23. Understanding genetic factors associated with virulence in L. monocytogenes will allow better detection and control of virulent strains. Sequence differences between L. monocytogenes strains HCC23 (non-virulent) and serotype 4b strain F2365 (virulent) were mapped and approximately 1,500 single nucleotide polymorphisms were resolved in HCC23. High throughput resequencing was conducted to resolve additional sequence differences. The total genome size of HCC23 was 2,943,866 bp in length. There were 74,923 high confidence mutations between F2365 and HCC23, indicating the two genomes are highly divergent. This indicates several genes are altered in HCC23 compared to virulent strain F2365, and several others are missing. When the genome of 4a strain HCC23 is completed, comparison of the genome of this avirulent strain with the F2365 genome will allow identification of novel virulence genes. Understanding genetic factors affecting virulence of L. monocytogenes will be important to improve food safety in seafood and other foods. This research directly addresses National Program 108 – Food Safety, Component 1.2 – Pathogens, Toxins and Chemical Contaminansts – Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Characterize genes that show genetic changes between virulent and avirulent L. monocytogenes. L. monocytogenes is a foodborne bacterial pathogen that employs a number of virulence-associated proteins (e.g., ActA and internalins) that enable penetration and movement within host cells. While actA and many internalin genes were examined previously by DNA arrays, they have not been fully characterized by PCR. Thus, oligonucleotide primers were designed from L. monocytogenes EGD-e actA and internalin genes (ie, lmo0327, lmo0331, lmo2396, lmo2445 and lmo2470) and used in PCR for examination of 29 L. monocytogenes and 5 other Listeria strains. L. monocytogenes serotypes demonstrated notable variations in their actA and internalin genes, with high virulence serotypes possessing more internalins than low virulence serotypes. Understanding genetic factors affecting virulence of L. monocytogenes will be important to improve food safety in seafood and other foods. This research directly addresses National Program 108 – Food Safety, Component 1.2 – Pathogens, Toxins and Chemical Contaminansts – Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Isolation of virulent and avirulent L. monocytogenes from the murine macrophage cell line J774.1. Understanding genetic factors associated with virulence in L. monocytogenes will allow better detection and control of virulent strains. Identifying differences in protein expression between virulent and avirulent strains will allow a better understanding of genetic determinants of virulence. However, studying of protein expression requires large amount of protein. By increasing incubation time and altering the infection procedure, we have increased the amount of bacteria harvested from J774 cells to enable us to harvest sufficient bacterial proteins to conduct high throughput proteomics of L. monocytogenes proteins. The differences in protein expression between virulent and avirulent strains at different time points may explain the difference in virulence. Understanding genetic factors affecting virulence of L. monocytogenes will be important to improve food safety in seafood and other foods. This research directly addresses National Program 108 – Food Safety, Component 1.2 – Pathogens, Toxins and Chemical Contaminansts – Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Development of a multiplex PCR for species- and virulence-specific determination of L. monocytogenes. L. monocytogenes is composed of multiple serotypes/strains showing varied virulence potential. As a means to implement effective control and prevention measures for L. monocytogenes, it is necessary to identify virulent, disease-causing strains from avirulent, nonpathogenic strains that are relatively harmless. After examination of a collection of 29 L. monocytogenes and 5 strains from the other Listeria species by PCR and Southern blot, a multiplex PCR was developed that incorporates genes (inlA, inlC, and inlJ) potentially related to virulence in L. monocytogenes gene. The species identity of the 29 L. monocytogenes strains was verified through the amplification of a 800 bp fragment with the inlA gene primers, and the virulence potential of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ gene primers, respectively. Use of the multiplex PCR targeting inlA, inlC, and inlJ genes facilitated rapid, simultaneous confirmation of L. monocytogenes species identity and virulence potential. Rapid, accurate identification of L. monocytogenes species and potential for virulence has important implications for controlling this important food pathogen. This research directly addresses National Program 108 – Food Safety, Component 1.2 – Pathogens, Toxins and Chemical Contaminansts – Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Evaluation of acid, alkali and salt tolerance among Listeria species. Being ubiquitously present in the natural environment, Listeria species (especially L. monocytogenes) are renowned for their ability to withstand external pH, temperature, and osmotic stresses, and to survive a variety of food manufacturing processes. Therefore, a detailed understanding of non-monocytogenes Listeria species in terms of their biochemical and molecular features is vital to the development of novel diagnostic procedures for improved discrimination of pathogenic from non-pathogenic Listeria species. We determined effect of extreme pH and salt concentrations on survival of 16 Listeria strains (with three each of L. grayi, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri, and one L. monocytogenes strain). There were significant differences among Listeria species in their ability to survive salt concentrations and pH. We are currently assessing if the differnces in tolerance of Listeria species and strains to salt concentration and pH are associated with their virulence. Understanding relationships between virulence and environmental tolerance in Listeria species will provide insights into virulence of this important pathogen and potential methods for controlling it. This research directly addresses National Program 108 – Food Safety, Component 1.2 – Pathogens, Toxins and Chemical Contaminansts – Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Determine effects of chemical compounds, diffusion method, and cooking method on masking of off-flavor compounds in fish. The incidence of ‘off-flavor’ remains a major issue for the catfish farming industry and other farmed seafood. Off-flavor in catfish is typically due to uptake of algal derived compounds which impart a objectionable flavor and odor to the fillet. Methods for reducing or masking off-flavor compounds would benefit the catfish farming industry. We determined effects of chemical compounds, diffusion methods, and cooking methods for reducing or masking off-flavor from 2-methylisoborneol (MIB) in processed channel catfish. There were no differences among diffusion methods (dipping, tumbling, injecting). Acetic acid (1-2%) reduced off-flavor odor, but induced a sour flavor. Citric or lactic acid at 1%, 3 ppm ozone for 10-30 min, 0.5% hydrogen peroxide for 20 min or, sodium bicarbonate at 1-7% were not effective for masking or reducing MIB. Sodas like 7-up® and Sprite® were shown to decrease off-flavor while adding a pleasant sweet flavor to the catfish fillet. Frying and marinating prior to baking were found to be effective in decreasing sensory off-flavor. GC/MS analysis showed that MIB was reduced by acetic acid and cooking but not by the soda treatments. Reduction or masking of off-flavor due to MIB appears to be possible and could greatly benefit the catfish industry by allowing sale of catfish that would otherwise have poor consumer acceptance. This research directly addresses National Program 106- Aquaculture; Component 9 Quality, Safety, and Variety of Aquaculture Products for Consumers. Determine effects of sampling site on level of off-flavor compounds in catfish flesh. Off-flavor compounds may not be consistent throughout catfish fillets, and therefore, the location on the fillet from which a sample is taken may influence if a sample is determined to be off-flavor. We evaluated effects of sampling location on off-flavor assessment in catfish fillets. Muscle in the peritoneal cavity contained 3-5X higher amount of geosmin than other parts. Other sites on the fillet showed differences in concentration of off-flavor, with lean areas showing less off-flavor than fatty areas. This suggests that fatty areas of the fillet, such as the muscle in the peritoneal cavity, should be sampled when screening for off-flavor catfish to insure a rigorous testing and minimize the incidence of truly off-flavor fish being classified as on-flavor. This research directly addresses National Program 106- Aquaculture; Component 9 Quality, Safety, and Variety of Aquaculture Products for Consumers.
5. Significant Activities that Support Special Target Populations
Assuring safety and improving quality of domestic farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. The USDA Census of Aquaculture in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 515 farms (38% of the total) reported annual revenues of less than $25,000. Expansion of markets through development of safe, high quality domestic catfish products will benefit U.S. catfish farmers, a significant portion of which are classified as small farmers.