DEVELOPMENT OF RAPID METHODS FOR DETECTION AND ENUMERATION OF VIBRIO VULNIFICUS, VIBRIO PARAHAEMOLYTICUS AND ENTERIC VIRUSES IN SEAFOOD AND HARVESTING AREAS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0220413
• Project No.: LAB94010
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2014

Project Director
Janes, M.

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
School of Nutrition and Food Sciences

Non Technical Summary
Economic Impact on Louisiana Oyster Industry: Vibrio vulnificus and V. parahaemolyticus continues to harass the Louisiana oyster industry because it is found in the marine environment and its ability to cause disease in certain consumers. Research efforts are in progress to develop technologies to eliminate or greatly reduce the V. vulnificus and V. parahaemolyticus burden in oysters. To complement these efforts, and permit investigators to assess their endeavors, methods are needed which can easily detect and enumerate V. vulnificus and V. parahaemolyticus cells by direct examination of shellfish. Our research project will develop rapid antibody methods that can be used by scientists, seafood processors and regulatory agencies. There is a continuing strong governmental and commercial demand for development of less costly, user-friendly antibody-based assays specific for pathogenic V. vulnificus and V. parahaemolyticus in raw oysters and other seafood products. Since Louisiana is one of the leading states in oyster harvesting, benefits should accrue to Louisiana and sister states on the Gulf of Mexico. Large multistate outbreaks of Norwalk virus in 1993 and Hepatitis A virus in 2005 were traced to consumption of oysters obtained from Louisiana harvesting sites. These outbreaks further highlighted the fact that there is very little data on virus occurrence in seawater. The lack of data is due to the absence of efficient methods that enable rapid detection of viruses. Most current methods are labor or time intensive, expensive, or impractical. Therefore, there is an acute need for the development of technologies that enable efficient detection of viruses in seawater samples. Our project will be to optimize methods for extraction of enteric viruses from large volumes of seawater, develop DNA methods for enumeration and detection of enteric viruses from seawater and assess the reliability of FRNA phages as indicators for Hepatitis A and/or Norovirus in shellfish harvests areas. FRNA phages are reliable indicators of the possible presence of human enteric viruses in seafood and seafood growing waters because they behave like water-borne viruses. Results from this project could lead to development of methods that can be used to monitor enteric viruses in seawater.

Animal Health Component

0%

Research Effort Categories

Basic

(N/A)

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	0811	1040	25%
712	4010	1090	25%
712	4010	1100	25%
712	4030	1101	25%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria; 0811 - Shellfish; 4030 - Viruses;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1101 - Virology; 1100 - Bacteriology;

Keywords

immunomagnetic separation

immunoblot

vibrio vulnificus

vibrio parahaemolyticus

antibodies

frna phage

hepatitis a

norovirus

Goals / Objectives
In the United States, seafood accounts for 26.5% of foodborne outbreaks. A majority of these cases are due to consumption of contaminated raw bivalve molluscan shellfish. Two of the major bacterial pathogens associated with raw oysters are Vibrio vulnificus and Vibrio parahaemolyticus. Because of the rapid progression and high mortality rates of V. vulnificus infections in humans, especially those victims with underlying chronic disease, gulf coast oysters are being scrutinized for the presence and level of this organism. Conventional methods for V. vulnificus, V. parahaemolyticus and enteric virus detection in raw oysters or other seafood involves lengthy selective cultural enrichment. Thus, the recovery, identification and enumeration of V. vulnificus, V. parahaemolyticus and enteric viruses in oysters or seawater can be overwhelming in logistics, materials and time, while the expeditious and specific identification of V. vulnificus, V. parahaemolyticus and enteric viruses in the laboratory is desirable. In addition, the need for user-friendly methods for generating quantitative data from raw oysters for HACCP and food and drug administration (FDA) regulatory purposes is another serious criterion for developing alternative methods for V. vulnificus, V. parahaemolyticus and enteric virus enumeration. The specific objectives for enumeration and identification of pathogenic Vibrios in oysters are as follows; produce species specific antibodies against pathogenic and/or total Vibrio parahaemolyticus and Vibrio vulnificus, produce monoclonal antibodies reactive with and specific for the thermostable direct (TDH) and TDH-related hemolysins and the H antigen(s) displayed on V. parahaemolyticus polar flagella, develop a direct colony immunoblot assay for detection of V. parahaemolyticus using anti-V. parahaemolyticus H and anti-hemolysin monoclonal antibodies, employ the colony immunoblot assay to detect known clinical and environmental V. parahaemolyticus isolates and examine any cross-reactions with other Vibrio species, employ the colony immunoblot assay to detect and enumerate environmental V. parahaemolyticus isolates in raw oysters harvested from the Gulf of Mexico over a 1 year period, and develop sensitive IMS (immunomagnetic separation) /DNA hybridization-based, IMS/colony Immunoblot and/or IMS/qPCR-based assays to detect Vibrio parahaemolyticus virulent and non-virulent strains and Vibrio vulnificus in oyster homogenates and seawater. The final goal of this research project will be to optimize qRT-PCR methods for detection and quantification of FRNA phages, Hepatitis A and Noroviruses in seawater samples and to demonstrate the integrated sampling and test methods to assess the reliability of FRNA phages as indicators for HAV and/or NV in shellfish harvests areas.

Project Methods
Monoclonal antibodies reactive with V. parahaemolyticus TDH/TRH and H antigens will be developed by previously described methods. Once the monoclonal antibodies for the species-specific hemolysins and H antigens are developed, then adaptation and optimization of the DCI (direct colony immunoblot) method for the detection of total and pathogenic V. parahaemolyticus isolates on selective and non-selective agar plates will be conducted. Next the specificity of the assay will be determined using other Vibrio species and pathogenic and non-pathogenic V. parahaemolyticus strains in mixed cultures on spread plates employing duplicate immunoblots. Finally, the assay will be used to detect and enumerate total and pathogenic V. parahaemolyticus isolates in seeded and unseeded oyster homogenates on selective agar plates in a 24-hour time frame. The V. parahaemolyticus DCI assay will be compared to the DNAH (DNA hybridization) method over a one year period following ISSC methods for a single laboratory validation of a new analytical method. The next stage of the investigation will be dedicated to the adaptation and optimization of existing IMS (Immunomagnetic separation) methods to isolate and concentrate V. vulnificus isolates in pure and then mixed cultures. Next, quantification of V. vulnificus in spiked oyster homogenates, with and without IMS, will be determined by established qPCR methods. The efficiency of the IMS/qPCR procedure will be compared to the Food and Drug Administration's DNA probe hybridization method. If good correlation between the two methods (IMS/qPCR and DNA probe hybridization) is observed, then the recovery and enumeration of environmental V. vulnificus isolates from freshly harvested oysters by IMS/qPCR will be determined every three months for one year. To assess the reliability of FRNA phages as indicators for HAV and/or NV in seawater collected from shellfish growing areas, seawater samples will be taken from 6 locations in the Gulf of Mexico over a two year period. Seawater samples will be collected in sterile 10 liter carboys. During each visit, a one liter sample of seawater will be obtained and labeled with the collector's initials, a unique sample identification number, time of sampling, date, and location. All sample containers will be capped, labeled, and stored in a sample cooler that had been previously chilled to 4degreeC. Samples will be immediately transported to the laboratory for analyses. All samples will be subjected to the filtration/anion exchange resin capture method and RNA will be extracted from enteric viruses attached to the resin beads and the presence of FRNA phages, HAV and NV will be determined by qRT-PCR assays. If positive HAV and NV are detected in the environmental samples they will be sent to a commercial lab for cloning and sequencing. Results from this research project will allow us to develop rapid enteric viral indicator methods that correlate to the presence of Hepatitis A and/or Noroviruses in shellfish growing areas.

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: Federal and State agencies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A graduate student is involved with the research How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Enteric virus-contaminated shellfish represents a significant health threat to shellfish consumers as well as an economic threat to the seafood industry. Male specific bacteriophage MS2 has been identified as a suitable indicator for water-borne enteric virus outbreaks, and its presence indicates a recent contamination. There is an urgent need for the development of rapid detection by federal and state agencies. In an attempt to improve the visibility and consistency of the current plaque assay, a spread plate technique was introduced instead of the pour plate technique used commonly in existing methods. Other parameters that influence the outcome of the plaque assay were also compared. Using thespread plate technique resulted in an increase of plaque size by approximately 50% and contributed to a better visibility. The addition of supplements (glucose, CaCl2 and thiamine)as well as the reduction of agar thickness and hardness also contributed to enhanced plaque visibility and increased plaque count. Among all the conditions tested, a supplemented, thin-bottom agar (10 ml 1% agar) and a supplemented, thin-top agar (10 ml 0.45% agar) with the spread plate technique gave the maximum countable plaques with a minimum standard deviation. When compared to other methods, it produced significantly higher plaque counts and lower variation.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Jiemin C., M. Janes. 2014. A double layer plaque assay using spread plate technique for enumeration of bacteriophage MS2. J. Virology Methods, 196:86-92.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Norovirus-contaminated shellfish represents a significant health threat to shellfish consumers as well as an economic threat to the seafood industry. There is an urgent need for the development of rapid concentration methods that enable efficient detection of viruses in seawater samples. Zeolite has the potential to be used in large scale concentration device for detection of enteric viruses in seawater. Our research project will help feel this gap. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The results of this project were presented at the IAFP meeting in 2013 in Charlotte NC as follows Jeimin Cormier and Marlene E. Janes. 2013. Extraction of hepatitis A virus from seawater using zeolite (213D, Tuesday 30, 2013). What do you plan to do during the next reporting period to accomplish the goals? 1) develop a concentration/elution method in combination with real-time PCR (qPCR) for detection of norovirus from seawater using zeolite

Impacts
What was accomplished under these goals? Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis, and outbreaks caused by this virus often occur due to fecal polluted waters. Rapid concentration and detection of viral contamination in water environments can prevent large scale economic loss and can identify the source of contamination within a short time. However, conventional methods for virus concentration are often laborious, time consuming, and subject to blockage issues. Furthermore, most methods require a secondary concentration step to reduce the final volume of samples. We developed a concentration/elution method in combination with real-time PCR (qPCR) for detection of HAV from seawater using zeolite. In the method, seawater was spiked with HAV and the viruses were extracted by zeolite. The viruses were then eluted with sodium dodecyl sulfate and detected via qPCR. Additionally, we modified the thermoconditions of PCR and reduced the time to 30 min, as opposed to 2 h for qPCR. Zeolite was able to extract virus particles from seawater with ~99% efficiency in less than 5 min and remained efficient in large volumes of seawater (10 L). The entire concentration/elution method was done in approximately 2 h and was at least 50 times more sensitive than direct detection of virus in seawater. Furthermore, compared to existing methods, this method eliminated the need for secondary concentration, and would be a low cost and a simple procedure.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Jiemin Cormier and Marlene Janes. 2013. Concentration of enteric virus indicator from seawater using granular activated carbon (Journal of Virology Methods, Accepted)
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Jeimin Cormier and Marlene E. Janes. 2013. Extraction of hepatitis A virus from seawater using zeolite (213D, Tuesday 30, 2013)

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The results of this project were presented at the IAFP meeting in Providence, Rhode Island, and the 4th Trans-Atlantic Fisheries Technology Conference in Clearwater Beach, Fl. PARTICIPANTS: M.E. Janes (PI) and J. Cormier, LSU AgCenter. TARGET AUDIENCES: Target audiences include microbiologists, food and aquaculture scientists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The use of activated carbon has been tested for extraction of MS2 bacteriophage from seawater and has the potential to be developed into a rapid concentration and detection method for enteric water-borne viruses. The effects of pH, salinity and contact temperature on the absorbance efficiency of activated carbon were investigated in artificial seawater. 108-109 PFU of MS2 bacteriophage and 1 g of activated carbon were inoculated into 500 ml of artificial seawater. After 3 h of constant stirring, activated carbon was separated from the seawater, incubated with 1 ml of trypsin-EDTA solution for 2 h at room temperature to release MS2 bacteriophage from the activated carbon, and RNA was extracted. qRT-PCR was conducted to determine the PFU of MS2 bacteriophage released from the activated carbon. Results indicated that warmer temperature provides significantly better efficiency (P<0.01) for activated carbon, and pH has no significant effect on the absorbance efficiency. Activated carbon has significant higher absorbance efficiency in seawater of salinities 10 ppt, 20 ppt and 40 ppt at 37degreeC (P<0.01). In 20 ppt seawater at 37degreeC, 1.0 g of activated carbon was able to absorb up to 2.5 times 106 PFU of MS2 from 109 PFU MS2 inoculated in the 500 ml seawater. Since shellfish harvesting area is usually around 20 ppt seawater, activated carbon can efficiently concentrate enteric viruses and assist the monitoring of enteric virus outbreaks.

Publications

• Jiemin Cormier, Miguel Gutierrez, Lawrence Goodridge, Marlene Janes. 2012 Extraction of enteric virus indicator from seawater using activated carbon. Presented at the IAFP meeting in Providence, Rhode Island, July 22 through 25 (Abstract number P3-04).
• Jiemin Cormier, Miguel Gutierrez, Lawrence Goodridge, Marlene Janes. 2012 Extraction of Enteric Virus Indicators from Seawater Using Activated Carbon. Presented at the 4th Trans-Atlantic Fisheries Technology Conference in Clearwater Beach, Fl. Oct. 29 through Nov. 2.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Two journal articles and one presentation at a national meeting were used to dessiminate project information. PARTICIPANTS: M.E. Janes, (PI), and R. Jadeja, LSU AgCenter. TARGET AUDIENCES: The oyster industry needs a rapid method for detection of Vibrio vulnificus in oysters. Our research project will help fill this gap. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Vibrio vulnificus (V. vulnificus) is considered to be the most invasive Vibrio in the U.S., causing high fatalities especially in immunocompromised individuals. The rapid detection of V. vulnficus is necessary for monitoring this pathogen in the environment and in seafood as a pro-active measure to reduce V. vulnificus-related infections. The purpose of this study was to develop a rapid, user friendly and compact screening dipstick device utilizing V. vulnificus anti H monoclonal antibodies, which can detect the presence of V. vulnificus from oyster homogenate within 5 min. The dipstick test strips were prepared by conjugating the species-specific anti V. vulnificus H monoclonal antibody 3-D-10 colloidal gold particles. Resultant antibody conjugate was dispensed onto a membrane at a rate of 1 micro-liter/cm. The control line was prepared by using Goat anti Mouse IgG and sprayed on the membrane at the rate of 1.5 micro-liter/cm. Specificity of the dipstick device was tested against pure culture of 8 vibrio strains while sensitivity was tested by utilizing serially diluted overnight grown V. vulnificus culture (106 to 101 CFU/ml). Sensitivity of the device was tested with spiked oyster meat homogenate (10g oyster meat + 20ml APW), spiked with V. vulnificus ATCC 27562, to reach concentrations from 101 to 106 CFU/ml. One ml from each sample was collect every hr for 6 hr and immediately tested with the dipstick. Our dipstick device successfully identified V. vulnificus and did not produce visible signal, for other Vibrio strains tested within 5 min. The lowest concentration of V. vulnificus that produced positive test strip results was 104 CFU/ml, and when combined with 5 hr enrichment, the sensitivity of the dipstick increased to 101 CFU/ml. Our dipstick assay could be an answer to seafood industries rapid pathogen detection needs.

Publications

• Qureshi, M. E. Janes and D. Hayes. 2011. Biocompatible/Bioabsorbable silver nanocomposite coatings. Journal of Applied Polymer Science 120:3042-3053.
• M. R. Cole, M. Li, B. El-Zahab, M. E. Janes, D. Hayes, and I. M. Warner. 2011. Design, Synthesis, and Biological Evaluation of β-Lactam Antibiotic-Based Imidazolium- and Pyridinium-Type Ionic Liquids. Chemical Biology & Drug Design 78:33-41.
• Jadeja, R., M.E. Janes, and J. Simonson. 2011. Dipstick assay for Vibrio vulnifus. IAFP, Milwaukee, WI. Abstract P3-07.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The results of this project were presented at the IAFP meeting in 2010 in Anaheim CA. In addition, one refereed journal article was generated. PARTICIPANTS: This research project fully supported one Ph. D. student Ravi Jadeja who graduated in December 2010. Dr. Janes was the lead PI on the project and assisted with the design of the research project, supervision of the student, and analysis of the data. TARGET AUDIENCES: There is a need by the oyster industry for the development of thermal treatment standard procedures and parameters in order to pasteurize the oyster meat without sacrificing the delicacy. Our research project will help fill this gap. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Oysters from the estuarine environments are well known to be carriers of Vibrio species. Due to the foodborne illnesses caused by consuming raw oysters, the FDA may require all raw oysters to have a post-harvest treatment to reduce Vibrio species. The current study was conducted to development a post-harvest treatment to reduce the levels of Vibrio vulnificus and Vibrio parahaemolyticus in shucked oysters by using low temperature treatments. Shucked oysters (300 g) were collected in sterile Whirl-Pak bags and spiked with V. vulnificus or V. parahaemolyticus to achieve about 1,000,000 CFU/g. Inoculated oyster meat samples were placed in water baths for 12 min at four different temperatures 40, 45, 50 or 55degreeC. After heating, oyster samples were rapidly cooled by placing the oyster meat on ice and immediately storing at 4degreeC. The following samples were analyzed for bacterial counts for all the four temperature treatments, inoculated oyster samples or control, heat-treated inoculated oysters without cooling, inoculated oysters heated then cooled on ice. Bacterial counts were determined at day 0, 1 and 2 by plating on nutrient agar with 2% NaCl, incubating plates at 37degreeC for 24 h and calculating CFU/g. V. vulificus was more sensitive to the temperature treatments reaching non-detectable levels at 50degreeC on day 0, 1 and 2. V. parahaemolyticus was more heat resistant at 50degreeC with average bacterial counts of 2.26 X 100 CFU/g after 1 day at 4degreeC. The most effective treatment was 55degreeC which reduced the bacterial counts for both Vibrio species to non-detectable on day 0, 1 and 2. Application of low heat treatment to shucked oyster meat can be a promising post-harvest treatment for control of Vibrio species in oysters.

Publications

• R. Jadeja, M.E. Janes, and J.G. Simonson. 2010. Immunomagnetic Separation of Vibrio vulnificus with Anti-Flagellar Monoclonal Antibody. Journal of Food Protection 73:1288-1293.
• R. Jadeja and M.E. Janes. 2010. Low-temperature treatment to reduce the risk of Vibrio species in shucked oyster. Presented at the IAFP meeting in Anaheim CA, July 31- August 4 (Abstract number P1-65).