SEROLOGY 101

Or How To Interpret Those Funny-Looking Graphs

Dr. Victoria Bowes Animal Health Centre Abbotsford, BC Presented June 4, 2003

Avian Immune System

NON-SPECIFIC and SPECIFIC border defenses (innate) surveillance (white blood cells) rapid elimination of harmful substances

Avian Immune System

NON-SPECIFIC IMMUNITY: anatomic skin, mucus, cilia body temperature genetic factors sneeze, cough, vomit age, nutrition

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

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

The Immune Response

antibody: a protein produced in response to the presence of a specific antigen antigen: a molecule capable of eliciting an immune response, usually large, complex and foreign

The Immune Response

Example:

An IB virus that has its surface receptors coated in antibodies cannot attach itself to the cells lining the trachea, the first step in infection.

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

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)

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

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

Blood Serum

The Animal Health Monitoring Lab

ELISA
E nzyme L inked I mmuno S orbant A ssay

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

The Elisa Plate Reader

ELISA plate

HI plate

Serological Strategies
DIAGNOSTIC: field challenge paired samples ASSESS VACCINATION RESPONSE: protective and maternal titres

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)

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

Limitations
seroconversion takes 4-20 days tests only circulating antibody static measure (rising, stable, falling) cant differentiate field vs. vaccine strains cross-reactions test error

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 the antibody of 64 2X the antibody of 16

Doubling Log Titres Dilutions 1 0 2 1 4 2 8 3 16 4 32 5 64 6 128 7 256 8 512 9 1024 10 2048 11

Interpretation No immunity Maternal Immunity Live Prime Vaccine Poor Immunity 2nd Live Prime Vaccine

Protection Against Mortality

Field Challenge

Prime Plus Killed

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

Interpreting Results
MEAN TITRES:

the average titre number (zero to >10,000)

each titre group (bottom line of graph) represents a different titre range depending on the virus

Titre Group 0 1 2 3 4 5 6 7

Reovirus MG/MS 0-537 538-1075

IB, IBD NDV 0-395 396-999

AE 0-444 445-769 770-1215

1076-1999 1000-1999

2000-2999 2000-2999 1216-1661 3000-3999 3000-3999 1662-2107 4000-4999 4000-4999 2108-2553 5000-5999 5000-5999 2554-2999 6000-7999 6000-7999 3000-3444

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

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

Interpretation

EXAMPLES
The following are typical histograms

5 week unvaccinated broilers negative titres

5 week broilers IBD vaccination or challenge?

5 week vaccinated broilers IBD challenge, poor vaccine?

62 week layer bloods waning titres

pre & post-killed IBD titres

pre & post-killed reovirus titres + challenge?

NDV titres poor prime boosted by killed vaccine

IBV uneven prime, suboptimal titres

Reovirus titres possible vaccine + field challenge?