PREVALENCE OF Kudoa thyrsites IN PACIFIC HAKE (Merluccius productus) AND THERMAL RESISTANCE OF Kudoa thyrsites AND Kudoa paniformis SPORES by GRACE TAMKEE B. Sc. (Agr.), The University of British Columbia, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Food Science Programme). We accepUhis thesis as conforming tO/theVequired standard THE UNIVERSITY OF BRITISH COLUMBIA October 2003 © Grace Tamkee, 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date QdfllflJg^ (ffi , 200^ DE-6 (2/88) Abstract Experiments were conducted to determine the potential of Pacific hake (Merluccius productus) fish meal to act as a vector of Kudoa thyrsites transmission to farmed Atlantic salmon (Salmo salar). This was done by determining the prevalence and intensity of K. thyrsites in Pacific hake destined for fish meal processing, developing a viability test for K. thyrsites, and determining the thermal resistance of K. thyrsites. Thermal resistance experiments were also performed for K. paniformis. Pacific hake samples were collected from the rendering plant in 2001 and 2002. The prevalence of K. thyrsites in the 2001 samples was 78.0%. Of these infections, 36.4% were light and 63.6% were moderate. The 2002 samples had a K. thyrsites prevalence of 80.1% infection, with 24.3% being light and 75.7% being moderate. Dyes used to test viability of other myxosporean parasites were examined for their suitability for K. thyrsites and K. paniformis. Methylene blue, fluorescein diacetate, and propidium iodide were suitable as a potential indicator of K. thyrsites viability. Only methylene blue was tested for K. paniformis, and it was found to be suitable. The decimal reduction times (D-value) of K. paniformis and K. thyrsites were determined using methylene blue as a viability test. The average D-value of K. paniformis in phosphate buffered saline (pH 7.0) was 18 hours at 53°C, while at 62°C and 69°C the average D-value was 4.5 hours and 0.80 hours, respectively. The z-value was 12C°. The average D-value for K. thyrsites at 43°C was 3.0 hours, and at 52°C and 60°C the average D-value was 1.4 hours and 0.030 hours, respectively. The z-value for K. thyrsites was 8.9C0. Given the available data, the likelihood of K. thyrsites surviving both fish meal and fish feed manufacturing are very low. ii Table of Contents Abstract ii Table of Contents ....iii List of Tables v List of Figures vi List of Tables - Appendix vii Acknowledgements viii 1.0 Introduction 1 2.0 Literature Review 3 2.1 Myxosporea and Kudoa..... 3 2.2 Kudoa and Pacific Hake 6 2.3 Fish Meal in Farmed Salmon Diet. 7 2.4 Kudoa and Atlantic Salmon 7 2.5 Heat Resistance and Measuring Viability 11 2.6 Summary 16 3.0 Materials and Methods 17 3.1 Prevalence and Intensity of K. thyrsites Infections 17 3.2 Trypsin Digestion to Release Kudoa Spores from Fish Meal 18 3.3 Isolation of Kudoa Spores from Fish Flesh 19 3.4 Enumerating Kudoa Spores 21 3.5 Development of a Viability Test for Kudoa Spores 22 3.5.1 Trypan Blue 22 3.5.2 Methylene Blue 22 3.5.3 Fluorescein Diacetate (FDA) and Propidium Iodide (PI) 23 3.6 Heat Resistance Determinations 24 3.6.1 Decimal Reduction Time (D-value) of Kudoa Spores 24 3.6.2 Calculation of Kinetic Parameter 25 3.7 Examination of Fish Oil as a Source of Kudoa Spores 26 4.0 Results and Discussion 27 4.1 Prevalence and Intensity of K. thyrsites Infections 27 4.2 Trypsin Digestion to Release Kudoa Spores 30 iii 4.3 Development of a Viability Test for Kudoa Spores 33 4.4 Heat Resistance Determinations 40 5.0 Conclusion 56 References 57 Appendix 67 iv List of Tables Table 1. Prevalence and intensity of K. thyrsites infections in Pacific hake collected in 2001 28 Table 2. Prevalence and intensity of K. thyrsites infections in Pacific hake collected in 2002 29 Table 3. Intensity of K. thyrsites infections in whole Pacific hake collected in 2001 and 2002 (n=50) 31 Table 4. Decimal reduction times (D-value) of K. paniformis 41 Table 5. Decimal reduction times (D-value) of K. thyrsites 42 v List of Figures Figure 1. K. thyrsites (formalin-treated) spores stained with methylene blue 35 Figure 2. K. thrysites spores stained with fluoroscein diacetate 36 Figure 3. K. thyrsites spores stained with propidium iodide. One spore is stained with fluorescien diacetate 37 Figure 4. K. thyrsites polar capsules fluorescing green, and the remainder of the spore fluorescing red 38 Figure 5. Survivor curve at 53°C for K. paniformis in phosphate buffered saline pH 7.0 43 Figure 6. Survivor curve at 62°C for K. paniformis in phosphate buffered saline pH 7.0 44 Figure 7. Survivor curve at 69°C for K. paniformis in phosphate buffered saline pH 7.0 45 Figure 8. Thermal death temperature sensitivity curve for K. paniformis in phophate buffered saline pH 7.0 46 Figure 9. Survivor curve at 53°C for K. thyrsites in phosphate buffered saline pH7.0 47 Figure 10. Survivor curve at 62°C for K. thyrsites in phosphate buffered saline pH 7.0 48 Figure 11. Survivor curve at 69°C for K. thyrsites in phosphate buffered saline pH 7.0 49 Figure 12. Thermal death temperature sensitiviy curve for K. thyrsites in phosphate buffered saline pH 7.0 50 Figure 13. Flow chart of fish meal processing .". 52 vi List of Tables - Appendix Table 1. Change in pH of PBS and HEPES buffer after change from room temperature to 40°C 69 Table 2. Change in pH of PBS and HEPES buffer after change from room temperature to 50°C 70 Table 3. Change in pH of PBS and HEPES buffer after change from room temperature to 60°C 71 Table 4. Change in pH of PBS and HEPES buffer after change from room temperature to 70°C 72 Table 5. Change in pH of PBS and HEPES buffer after change from room temperature to 80°C 73 Table 6. Change in pH of PBS and HEPES buffer after change from room temperature to 90°C 74 vii Acknowledgements This thesis would not have been possible without the help of so many people. A special thanks to Keng Pee Ang, Judy Chan, Ron Da Silva, Jenny Dawson-Coates, Don, Doug Davidson, Danilo de Joya, Yolanda de Joya, Tim Durance, Christy Falkenberg, Paul Hardy-Smith, Rob Jones, Simon Jones, Nicholas Knott, Jason Mann, Peter McKenzie, Grant Saar, Chris Seaman, Brent Skura, Val Skura, Patrick Tamkee, Dave Whitaker, and Sherman Yee. Special acknowledgments for financial support are due to the Natural Sciences and Engineering Research Council, the Science Council of British Columbia, EWOS Canada, and West Coast Reduction. viii 1.0 Introduction Kudoa thyrsites is a myxosporean parasite that has been problematic on salmon farms in British Columbia due to their association with softening effect on muscle tissue of Atlantic salmon (Salmo salar) (Whitaker and Kent, 1991; Whitaker et al., 1994). Wild fish species can also be infected with K. thyrsites. Some of these hosts include Pacific halibut (Hippoglossus stenolepis), lingcod (Ophiodon elongates) and Pacific hake (Merluccius productus) (Moran et al., 1999b). Pacific hake is used to manufacture fish meal, which is subsequently used in the production of fish feed for farmed salmon. While it is known that Pacific hake can be infected with K. thyrsites, there are no current studies stating the level of infections. Therefore, Pacific hake that will be used for fish meal processing should be examined for the prevalence and intensity of K. thyrsites. In addition, it is unclear whether fish meal processing will inactivate K. thyrsites spores. Though the transmission of K thyrsites is thought to be indirect, the life cycle has not yet been discovered. Therefore, there may be potential for fish meal to act as a vector for parasite transmission. In effect, studies need to be undertaken to determine the possibility of K. thyrsites surviving fish meal manufacturing. Currently, there is no research available indicating whether temperatures and times used in the manufacturing of hake fish meal will eliminate K. thyrsites spores. At the same time, if the spores are not destroyed, fish feed manufacturers and salmon farmers fear that the fish meal may have the potential to act as a source of K. 1 thyrsites spores that may later infect farmed Atlantic salmon. If fish feed manufacturers could use Pacific hake fish meal for processing fish feed instead of importing fish meal from South America, it is estimated that fish feed manufacturers could save $1 million dollars per year (Mann, 2000). Therefore, it was hypothesized that Pacific hake would be infected with K. thyrsites and that elevated temperatures would render K. thyrsites and K. paniformis nonviable. Thus, fish meal would not act as a vector for K. thyrsites transmission. The main objective of the current study was to determine the possibility of fish meal to act as a vector for K.