Infectious Hematopoietic Necrosis Virus (IHNV) causes a significant proportion of disease in trout as well as in salmon. However, there is currently no commercial vaccine available to easily immunize fish against IHNV. A preventive treatment to reduce these considerable losses would be a significant contribution to the farmed salmonid industry. Bacteria belonging to the genera Pseudomonas, Achromobacter, coryneforms, Vibrio and Alteromonas sp. were shown to have anti IHNV properties. Given the encouraging data already existing on its antiviral potential, we have chosen to evaluate Pseudoalteromonas sp. as a probiotic for protection against IHNV disease in rainbow trout. OBJECTIVES: Develop unique antiviral probiotic based products that provide protection against viral infection in trout and salmon fish APPROACH: This project first involves the use of in-vitro methods to select for exhibition of anti viral properties by Pseudoalteromonas sp. bacteria. Results from these assays will allow selection of a best candidate for incorporation into a diet to be fed to rainbow trout before challenge with IHNV. Results from the challenge studies will assess the ability of the selected probiont protected in gastric resistant encapsulation form to protect and deliver the probiotic to the site of action as well as the capacity of the bacteria to exhibit in vivo anti viral activity PROGRESS: 2004/05 TO 2004/12 Task 1 Culture of P. undina: A selected strain of P. undina was cultured in a 100 liters fermenter using an optimized culture media and obtained over 2 kg of bacterial wet biomass. Detection and quantification of IHNV. EPC cells was inoculated with IHNV using a virus inoculum at 2.5x10EXP7 pfu/ ml. Tissue samples (liver, kidney, spleen, adipose tissue and pectoral fins) were collected and total RNA isolated from EPC cells, IHNV-infected and healthy fish. The IHNV primers were designed based on a published sequence of the virus (GenBank accession number L40883, IHNV reference strain WRAC). The SYBR Green real-time RT-PCR amplifications were performed in a Bio-Rad iCycler iQ TM (Bio-Rad Laboratories, Inc., Richmond, California). IHNV was successfully detected in liver, kidney, spleen, adipose tissue and pectoral fin. For both the laboratory-challenged and the filed samples. In-vitro Competitive Adhesion Assay: Gut and gill tissues of trout were exposed to P. undina at a concentration of 10EXP8/ml for 60 min and increasing levels of IHNV. IHNV G-gene and Beta-actin genes were detected by SYBR Green real-time RT-PCR. P. undina did not inhibit viral binding to gut or gill tissues of rainbow trout fingerling as assessed by the relative IHNV load. There was no significant correlation between the levels of viral inoculum and the levels of the virus detected at the end of competitive inhibition. The in vitro assay, therefore, suggests that P. undina did not inhibit the IHNV binding to the guts and gills of rainbow trout. Task 2 Undina encapsulation and Diet preparation: Undina biomass obtained from the 100 liters fermenter was encapsulated in a mixture of sodium alginate and high amylose maize. The resultant micro spheres were top-coated on a commercial start up diet for trout. Undina survival in microbounded diet: The undina load in the diet was established initially at 3.3X10EXP/g diet. This concentration load was maintained over the feeding period of the trout IHNV challenge experiments at Clear Spring. Trout IHNV challenge experiments: Eight groups of 25 rainbow trout fed the test or control diet (four replicates per diet) at about 4% of their body weight per day. challenges were scheduled for 2 and 3weeks post-feeding Virus challenge was performed by injecting each rainbow trout with IHNV. Dead fish were recorded over 28 days post injection. Conclusion: Survival of P.undina fed fish was not different from control fed fish (P- 0.05) in both IHNV challenge studies. We have concluded that P. undina diet did not provide any protection against IHNV infection in rainbow trout. Final conclusions Both in vitro adhesion and in vivo challenge studies failed to show any positive effect of P. undina against IHNV infection in rainbow trout. Although we have found that this probiotic can protect the fish against bacterial infections (V. anguillarum, S. Inae and A. salmonicida) as well as against viral infection in shrimp (WSSV), our present work found no protection against IHNV infection. We have successfully established a highly sensitive and quantitative assay for IHNV infected fish using a real-time RT-PCR assay and SYBR Green. IMPACT: 2004/05 TO 2004/12 Our main goal to develop a prventive treatment for IHNV infection in rainbow trout by using probiotic feed could not be accomlished through the present work. The probiotic bacteria (P. undina we have selected failed to provide any protection in both in vitro copetative adhesion studies and in vivo challenge experiments. We have however, successfully developed highly sensitive and realible method for detecting and quantifieng IHNV in infected fish. The SYBR Green real-time assay that was developed in this project is rapid, highly sensitive and reproducible. The ability to detect the virus in pectoral fins indicates the potential of this method in developing a non-invasive detection method for IHNV and perhaps other viruses infecting salmonids. In addition, a non-invasive tissue sampling will be very valuable for large scale virus screening of fish in aquaculture facilities as well as for epidemiological studies using SYBR Green real-time RT-PCR. PUBLICATIONS (not previously reported): 2004/05 TO 2004/12 Dhar, A.K., Bower, R., LaPatra, S., Licon, K.S., Read, B. and Harel. M. 2005. Detection of infectious hypodermal necrosis virus (IHNV) in trout by real-time reverse transcriptase-polymerase chain reaction (RT-PCR): Implications for developing non-invasive sampling for epidemiological studies. In final preparation for submission to Applied Environmental Microbiology. PROGRESS: 2004/05/15 TO 2004/12/31 Task 1 Culture of P. undina: A selected strain of P. undina was cultured in a 100 liters fermenter using an optimized culture media and obtained over 2 kg of bacterial wet biomass. Detection and quantification of IHNV. EPC cells was inoculated with IHNV using a virus inoculum at 2.5x10EXP7 pfu/ ml. Tissue samples (liver, kidney, spleen, adipose tissue and pectoral fins) were collected and total RNA isolated from EPC cells, IHNV-infected and healthy fish. The IHNV primers were designed based on a published sequence of the virus (GenBank accession number L40883, IHNV reference strain WRAC). The SYBR Green real-time RT-PCR amplifications were performed in a Bio-Rad iCycler iQ TM (Bio-Rad Laboratories, Inc., Richmond, California). IHNV was successfully detected in liver, kidney, spleen, adipose tissue and pectoral fin. For both the laboratory-challenged and the filed samples. In-vitro Competitive Adhesion Assay: Gut and gill tissues of trout were exposed to P. undina at a concentration of 10EXP8/ml for 60 min and increasing levels of IHNV. IHNV G-gene and Beta-actin genes were detected by SYBR Green real-time RT-PCR. P. undina did not inhibit viral binding to gut or gill tissues of rainbow trout fingerling as assessed by the relative IHNV load. There was no significant correlation between the levels of viral inoculum and the levels of the virus detected at the end of competitive inhibition. The in vitro assay, therefore, suggests that P. undina did not inhibit the IHNV binding to the guts and gills of rainbow trout. Task 2 Undina encapsulation and Diet preparation: Undina biomass obtained from the 100 liters fermenter was encapsulated in a mixture of sodium alginate and high amylose maize. The resultant micro spheres were top-coated on a commercial start up diet for trout. Undina survival in microbounded diet: The undina load in the diet was established initially at 3.3X10EXP/g diet. This concentration load was maintained over the feeding period of the trout IHNV challenge experiments at Clear Spring. Trout IHNV challenge experiments: Eight groups of 25 rainbow trout fed the test or control diet (four replicates per diet) at about 4% of their body weight per day. challenges were scheduled for 2 and 3weeks post-feeding Virus challenge was performed by injecting each rainbow trout with IHNV. Dead fish were recorded over 28 days post injection. Conclusion: Survival of P.undina fed fish was not different from control fed fish (P- 0.05) in both IHNV challenge studies. We have concluded that P. undina diet did not provide any protection against IHNV infection in rainbow trout. Final conclusions Both in vitro adhesion and in vivo challenge studies failed to show any positive effect of P. undina against IHNV infection in rainbow trout. Although we have found that this probiotic can protect the fish against bacterial infections (V. anguillarum, S. Inae and A. salmonicida) as well as against viral infection in shrimp (WSSV), our present work found no protection against IHNV infection. We have successfully established a highly sensitive and quantitative assay for IHNV infected fish using a real-time RT-PCR assay and SYBR Green. IMPACT: 2004/05/15 TO 2004/12/31 Our main goal to develop a prventive treatment for IHNV infection in rainbow trout by using probiotic feed could not be accomlished through the present work. The probiotic bacteria (P. undina we have selected failed to provide any protection in both in vitro copetative adhesion studies and in vivo challenge experiments. We have however, successfully developed highly sensitive and realible method for detecting and quantifieng IHNV in infected fish. The SYBR Green real-time assay that was developed in this project is rapid, highly sensitive and reproducible. The ability to detect the virus in pectoral fins indicates the potential of this method in developing a non-invasive detection method for IHNV and perhaps other viruses infecting salmonids. In addition, a non-invasive tissue sampling will be very valuable for large scale virus screening of fish in aquaculture facilities as well as for epidemiological studies using SYBR Green real-time RT-PCR. PUBLICATIONS: 2004/05/15 TO 2004/12/31 Dhar, A.K., Bower, R., LaPatra, S., Licon, K.S., Read, B. and Harel. M. 2005. Detection of infectious hypodermal necrosis virus (IHNV) in trout by real-time reverse transcriptase-polymerase chain reaction (RT-PCR): Implications for developing non-invasive sampling for epidemiological studies. In final preparation for submission to Applied Environmental Microbiology
