UNIVERSITY OF MINNESOTA Hamada. A. Aboubakr1,2, Sagar M. Goyal2 Antiviral Effects of Lactococcus lactis on Feline Calicivirus, A Human Norovirus Surrogate 1Department of Veterinary Population Medicine and Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA . 2Food Science and Technology Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt. UNIVERSITY OF MINNESOTA ALEXANDRIA UNIVERSITY Foodborne viruses, particularly human norovirus and hepatitis virus type A, are a cause of concern for public health and world economy. Hence, there is a need to explore novel and effective biological techniques for prevention of foodborne viral contamination, especially in minimally processed and ready-to-eat foods. This study aimed to determine the antiviral activity of a probiotic lactic acid bacterium against feline calicivirus (FCV), a surrogate of the human NV. Bacterial growth medium filtrate (BGMF) of Lactococcus lactis subsp. lactis LM0230 and its bacterial cell suspension (BCS) were evaluated for their antiviral activity against FCV grown in Crandell-Reese Feline Kidney (CRFK) cells. No significant antiviral effect was seen when cells were pre-treated with either BGMF (raw or pH 7-adjusted BGMF) or BCS. However, pre-treatment of the virus with BGMF and BCS resulted in a reduction in virus titer of 1.3 log10 TCID50 and 1.8 log10 TCID50, respectively. The highest decreases in FCV infectivity (7.5 and 6.0 log10 TCID50) were attained by co-treatment of FCV and CRFK cells with undiluted pH 7-adjusted BGMF and 100 µL of BCS containing 5.1×108 CFU/ml, respectively. These preliminary in vitro results indicate the possibility of using probiotics and lactic acid bacteria for bio-prevention of foodborne viral contamination. This finding needs validation by continuing studies on the role of probiotics and lactic acid bacteria in the inactivation of viruses in various types of foods. ABSTRACT Fig.1. Effect of cells pre-treatment with BGMF on FCV infectivity Fig.2. Effect of cells pre-treatment with BCS on FCV infectivity Fig.3. Effect of virus pre-treatment with BGMF Fig.4. Effect of virus pre-treatment with BCS (virus adsorption) Fig. 5. Co-treatment of cells with BGMF and virus Fig. 6. Co-treatment of cells with BCS and virus (competition assay) RESULTS Cytopathogenic Effect Cytotoxic Effect CRFK Normal Monolayer Table1. Cytotoxicity of BGMF to CRFK cells Dilution BGMF Raw pH 7 - adjusted + - 1/5 1/10 1/20 1/50 1/100 1/1000 + Cellular morphological alteration (cytotoxicity) was observed - No cellular morphological alteration was observed INTRODUCTION AIM OF INVESTEGATION Foodborne illnesses continue to be a significant concern not only threatens the public health but also the world economics. The mean economic cost of foodborne illness is approximately $152 billion in United States 1. To study the anti-viral activity of a probiotic lactic acid bacterium , Lactococcus lactis subsp. lactis LM0230, against feline calicivirus (FCV), as a surrogate of human NV. METHODS 570- 800 Deaths 56,000-71,000 Hospitalizations 400,000 Emergency Department Visits 1.7-1.9 Million Outpatient Visits 19-21 Million Total Norovirus Illnesses Annual Estimates of Norovirus Illnesses and Associated Outcomes Human NV is causing “winter-vomiting disease” or “stomach-flu” referring to their rapid spread in human populations especially during winter months. Enteric viruses, particularly human norovirus (NV), are the leading cause of foodborne illness in developed countries as epidemiological evidences indicate2. Bacterial growth medium pH-7 adjusted BGMF Raw BGMF without adjusting the pH Bacterial Cells 30 mL of MRS have been inoculated with Lactococcus lactic cells then incubated anaerobically for 24 h at 30 °C. Centrifugation 3,000 rpm for 15 min Preparation of BCS by suspending the pellets in 10 ml of sterilized peptone phosphate water broth Filter sterilization Adjust pH to 7 BGMF Bacterial Growth Medium Filtrate 1- Growing The Bacterium Freezing, cooling and mild heat treatments (minimal food processing) are not effective in significantly reducing virus titers 3. There is a real need to develop novel, efficient and safe strategies for controlling viral contamination in foods. Known Causes of Foodborne Illness Outbreaks, U.S. 2006-2010 CONCLUDING REMARKS REFERENCES 2- Treatments Lactococcus lactis subsp. lactis LM0230 had antiviral activity against FCV (Norovirus surrogate). Lactic acid bacteria and probiotics-based fermented food show promising applications in controlling viral food contamination. 1- Pre-treatment of CRFK cells with BGMF (Raw and pH-7 adjusted) 2- Pre-treatment of CRFK cells with BCS. 3- Pre-treatment of FCV with BGMF (Raw and pH-7 adjusted) 4- Pre-treatment of FCV with BCS. 5- Co-treatment of CRFK cells with BGMFs+FCV at the same time. 6- Co-treatment of CRFK cells with BCS+FCV at the same time. 1-Scharff R. L. (2010). Health-related costs from foodborne illness in the United States http://www.pewhealth.org/uploadedFiles/PHG/Content_Level_Pages/Reports/PSP-Scharff%20v9.pdf 2- The US Centers of Disease Control and Prevention CDC (2013) http://www.cdc.gov/norovirus/trends-outbreaks.html 3- Mormann S, Dabisch-Ruthe M, and Becker B. (2010). Inactivation of norovirus in foods. inoculation study using human norovirus. Fleischwirtschaft, 90:116–121 ANTI-VIRAL ACTIVITY of LACTIC ACID BACTERIA WAS HYPOTHESIZED. IT CAN BE USED AS BIOPRESERVATIVE FUTURE WORK CONTACT 3- Virus Titration 1- Study the mechanisms of LAB and probiotics antiviral effect. 2-Study LAB antiviral activity in food models. College of Veterinary Medicine, University of Minnesota. 1333 Gortner Ave St. Paul, MN 55108, USA. Cell Phone: 651-600-5627; Email: aboub006@umn.edu Using cytopathogenicity -based titration method (Kärber method) expressing the titer as Tissue Culture Infecting Dose (TCID50)