Understanding Avian Serology: Interpreting Immune Responses and Serological Testing

1. SEROLOGY 101 Or How To Interpret Those Funny-Looking Graphs Dr. Victoria Bowes Animal Health Centre Abbotsford, BC Presented June 4, 2003

2. Avian Immune System • NON-SPECIFIC and SPECIFIC • border defenses (innate) • surveillance (white blood cells) • rapid elimination of harmful substances

3. Avian Immune System • NON-SPECIFIC IMMUNITY: anatomic – skin, mucus, cilia body temperature genetic factors sneeze, cough, vomit age, nutrition

4. Avian Immune System • SPECIFIC IMMUNITY: • Passive (provided in the yolk to chicks) • Acquired (must be produced by the bird) (by infection or vaccination) • Cells – lymphocytes, macrophages • Antibodies - produced by B-lymphocytes • Memory

6. The Immune Response • Must recognize foreign or potentially harmful intruders • Produce and co-ordinate specific (antibodies) and non-specific (lymphocytes) reactions to ATTACH, TRAP and ELIMINATE

7. The Immune Response • antibody: a protein produced in response to the presence of a specificantigen • antigen: a molecule capable of eliciting an immune response, usually large, complex and foreign

10. The Immune Response • Example: An IB virus that has it’s surface receptors coated in antibodies cannot attach itself to the cells lining the trachea, the first step in infection.

11. The Immune Response • PRIMARY: novel foreign antigen is processed, specific antibodies are slowly produced (short-lived) and “memory cells” become dormant • SECONDARY: “memory cells” encounter their specific antigen and are activated, antibody production is rapid and potent

13. Vaccines • Vaccines = INSURANCE (something you may never use but you be very glad to have if ever needed) • “Live” : gives best immune response but has side effects (weakened strains of virus) • “Killed”: less side effects but poorer immune response (excluding adjuvants)

14. Serology • The detection of antibodies in serum • Test methods: plate agglutination hemagglutination inhibition ELISA • Interpreting results: negative vaccination field challenge

15. Blood • 55% fluid: mostly water • 45% solid: cells (500:1 red : white cells), enzymes, proteins, nutrients • transports oxygen & nutrients while removing waste • serum Vs plasma

16. Blood Serum

17. The Animal Health Monitoring Lab

19. ELISA E nzyme L inked I mmuno S orbant A ssay

20. ELISA • easily automated • sensitive & specific • antigen-coated test plates capture specific antibodies in diluted blood sample and reagents turn shades of blue depending and the amount of antibody present • generates computer graph “histogram”

21. The Elisa Plate Reader

22. ELISA plate

24. HI plate

26. Serological Strategies • DIAGNOSTIC: field challenge “paired” samples • ASSESS VACCINATION RESPONSE: “protective” and maternal titres

27. Serological Strategies • small sample size is used to infer the condition of a larger population of birds • statistical techniques dictate sample size of significance • variables: flock size 12-24 /air space? testing type • pooling samples reduces test sensitivity (dilutional)

28. Interpretation • Serology results must be interpreted in association with other sources of information: vaccination history production data condemnation data clinical signs necropsy findings

29. Limitations • seroconversion takes 4-20 days • tests only “circulating” antibody • static measure (rising, stable, falling) • can’t differentiate field vs. vaccine strains • cross-reactions • test error

30. Titres or Titers • a unit of measurement of antibody • uses pre-determined reference numbers • historically measured by sample dilution until a standard reaction was produced dilution = “titre” • a titre of 32 ½ the antibody of 64 2X the antibody of 16

34. Interpreting Results 123 A BB 18 X 345 – IBD-XR • 123 – flock # • A or B – 1st or 2nd bleeding • BB – broiler breeder (or TU, BR) • 18 – age in weeks • X – submitter code (hatchery, vet) • 345 – owner code • IBD-XR – test name

35. Interpreting Results • MEAN TITRES: the averagetitrenumber (zero to >10,000) • each titre group (bottom line of graph) represents a different titre range depending on the virus

37. Interpreting Results • UNIFORMITY: %CV (coefficient of variation) < 30% excellent 30-50% good 51-80% fair > 81% poor or bimodal

39. Standards • Optimum Titres: minimum titre level to provide long duration immunity for breeder hens, also assuming protective. Assessed 4 weeks post-killed vaccine. ND 8/10 > 7 IBV 8/10 > 6 IBD 8/10 > 8 Reo 8/10 >5 AE 8/10 >4

41. Interpretation EXAMPLES The following are typical histograms

42. 5 week unvaccinated broilersnegative titres

43. 5 week broilersIBD vaccination or challenge?

44. 5 week vaccinated broilersIBD challenge, poor vaccine?

45. 62 week layer bloods – waning titres

46. pre & post-killed IBD titres

47. pre & post-killed reovirus titres+ challenge?

48. NDV titres poor prime boosted by killed vaccine

49. IBVuneven prime, suboptimal titres

50. Reovirus titrespossible vaccine + field challenge?