What causes disease in fish?

1. Scott WilliamsARI Wet Lab ManagerFormer Brood Operations Manager / Genetics Research Clear Springs Foods

2. What causes disease in fish?

3. The Environmentcommercial trout culture • Clear Springs Foods Brood Division • Two facilities • Snake River / Research • Selection site • Brood propagation site • 5% of egg production • Soda Springs • Egg production facility

4. Improving the hostselection goals Primarily • Growth rate • Indirectly for feed conversion rate Secondarily • Disease resistance

5. Improving the Fish through Animal Breeding • Selecting the Parents of the Improved population • Using an Animal Model • Genetic merit based on: • Individual records • Family records

6. Family and Within Family Selection for Size at 273 Days • 100 to 110 full-sib families per generation • 10 families per generation from randomly bred line • Families selection: • Families with poor early survival were discarded • Families with high mean weights at 273 days were saved • Within the selected families: • pit tag top 15 to 20% of individuals

7. Selection for GrowthSize at 273 Days • 40 days Post Spawn, reduced to 600 individuals • 63 days PS, reduced to 100 individuals • 76 days PS, combined with another family in 100 gal tank • 161 days PS, each family was given unique clip/brand • 162 PS, moved to raceway • 273 PS, final measurement taken

8. Mean Weights at 328 Days PFodd year generation

9. Mean Weights at 273 Dayseven year generations

10. Correlated traits questions: • How does selection for rapid growth affect other traits? • Conformation? • Fillet fat levels? • Disease resistance?

11. Genetics questionsrelated to disease: • Is the ability to survive an IHNV infection a heritable trait, i.e., is it a trait that can be affected by selection? • How does IHNV survival correlate to rapid growth?

12. Relationship of growth to IHNV Susceptibility

13. Relationship of growth to IHNV Susceptibility

14. What is heritability? A measure of the degree to which the variance in the distribution of a phenotype is due to genetic causes. Or simply, the degree to which genes control expression of a trait.

15. How heritable is the IHNV survival trait? Estimate of the heritability is <0.83 This estimate confounds sources of genetic variation other than additive. This appears to be a highly heritable trait though the actual value is lower than this estimate.

16. Heritabilities and Genetic CorrelationsBetween Size and IHNV Susceptibility Fish with higher mean weights at the time of challenge, had greater CPM Groups with higher ending weights had sibling with higher CPM rates

17. Tagged families from the 2000 year classfamilies tagged based 1° on IHNV resistance

18. Are there other traits correlated to disease resistance that could be more easily measured? Parental Humoral Response humoral response - The immune response involving the transformation of B cells into plasma cells that produce and secrete antibodies to a specific antigen.

19. Traits Correlated to Disease Resistance:Parental Humoral Response • Parents of the selected fish were PIT tagged • Eight months prior to spawn • fish were IP exposed to attenuated form of the IHN virus. • Six months later months later • Blood samples were collected from broodstock. • Samples and tag ID were correlated • Samples were used to evaluate each fish’s ability to produce anti-IHNV neutralizing antibodies • Humoral response

20. Do the parental humoral responses correlate to the offspring challenge results? Weak effect of maternaltiter on offspring survival rates. Lower titer in the female associated with higher mortality rate in the offspring.

21. Improved Measurement of Disease Resistance At 63 days post spawn • Two challenges • Coldwater disease challenge • Two replicates of 50 fish each • Subcutaneous injection of Flavobacterium psychrophilum • IHNV challenge • One replicate of 50 fish • water borne exposure to IHNV in a closed, oxygenated system for 1 hour • 10,000 plaque forming units/ml • Volume of water that is 10 times the total weight of the fish

22. Improved Measurement of Disease Resistance • Mortality were collected and recorded daily • Necropsies were performed on a fraction of the mortality to verify that the challenge organism was the cause of death • Total cumulative percent mortality (CPM) and mean days to death (MDD) were calculated for each replicate

23. Distribution of Cold Water Disease Mortality 2001

24. Average CWD Mortality Rate by Strain

25. Distribution of IHNV Mortality Rates in 2001

26. Average mortality rate by strain

27. Distribution of CPM rates for Selected line 2002

28. Relationship between mortality rates 2001

29. Relationship between Mortality rates in 2001

30. Relationship between mortality rates 2002

31. Mortality rates within Control and Selected lines in 2002

32. Selection Program Modifications • 1º Family selection on disease resistance • Entire families were culled based on the mortality rates of replicates challenged • IHNV • Coldwater disease (CWD) • 2º Within family size • Saving the largest individuals within the families with the best survival rates

33. Family selection decision based on mortality index • Index based on mortality rates of both of the pathogens • Families with highest index scores were saved for grow-out phase of evaluation • All other families terminated after growth evaluation at 273 days

34. Assessing disease resistance &within family selection for size 273 days of age • Weigh every member of all remaining families • Return on day 274 • Pit tag largest 15% within each of these families

35. Directions for growth and disease resistance selection • Livability in the selected populations appeared to be decreasing relative to the unselected control line • Common occurrence for lines selected primarily for improved growth, e.g., poultry

36. Directions for growth and disease resistance selection • 1° selection based on a survival index • Index takes into account survival traits • 2° selection will be based on growth rate • Results: • Improved survival to IHNV • However, reduced rate of weight increases

37. Identification of DNA Markers Associated With IHNV Resistance in Rainbow Trout • Develop a rainbow trout linkage map • Find molecular markers linked to loci affecting resistance to infectious hematopoietic necrosis virus in rainbow trout • Develop a breeding plan to increase IHN resistance in rainbow trout using the genetic markers (Marker Assisted Selection)

38. Identification of DNA markers • Two strain of rainbows • One anadromous and with elevated resistance to IHNV • The other a domestic strain with good growth and low resistance to IHNV • Produce F1 cross • Back-crossing members of each family (F1) with rainbow trout produced an BC1 generation • Challenge with IHNV • Collect mortality and survivors • Use both AFLP & microsatellite markers • Look for polymorphisms associated with survival or mortality

39. Marker Assisted Selection for Trout Improvement Molecular tools GrowthDisease Resistance resistant susceptible large small DNAextraction QTLanalysis resistant large selective breeding large & resistant

40. Marker Assisted Selection Theoretically MAS has the potential to greatly accelerate genetic improvement livestock, plants and aquatic animals. Initial data from livestock have demonstrated that MAS can be more productive than traditional breeding, but some cases can be equally or less effective than selective breeding. Limited data from aquatic species.