1. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Diagnostic Methods for Avian Mycoplasmas Hendrik R. Froin

2. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Mycoplasma Diagnosis Diagnosis is based on the flock as a whole, not individual birds

3. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Criteria for Diagnosis Clinical signsClinical signs –Usually present in turkeys –Inconsistent in chickens Detection of antibodyDetection of antibody –Agglutination, HI, ELISA Detection of the organismDetection of the organism –Culture, PCR, immunohistochemistry

4. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Clinical Signs Respiratory signsRespiratory signs SynovitisSynovitis Egg production dropsEgg production drops Increased mortality?Increased mortality? Respiratory or leg problems in progenyRespiratory or leg problems in progeny

5. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Purpose of Serology Assist in diagnosis when signs are presentAssist in diagnosis when signs are present Screening of breeding flocks in control programsScreening of breeding flocks in control programs –Serology should help to identify flocks which warrant a closer look

6. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Utility of Serology Serology can be definitive if positive and associated with clinical signs or if a high % of the flock is positive with high HI or ELISA titersSerology can be definitive if positive and associated with clinical signs or if a high % of the flock is positive with high HI or ELISA titers In most situations positive or suspicious serology is confirmed by culture and/or PCRIn most situations positive or suspicious serology is confirmed by culture and/or PCR

7. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Sensitivity Ability of a test to detect an infected animal or flock (should not give false negative results)

8. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Specificity Ability of a test to detect birds or flocks which are negative (should not give false positive results)

9. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Characteristics of Agglutination Cheap and fastCheap and fast Detects primarily IgM (the first class of antibody produced)Detects primarily IgM (the first class of antibody produced) Sensitive (except in some cases of MS)Sensitive (except in some cases of MS) Lacks specificity (tends to give false positive results)Lacks specificity (tends to give false positive results)

10. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Characteristics of HI High degree of specificityHigh degree of specificity Low to medium sensitivityLow to medium sensitivity Detects primarily IgGDetects primarily IgG Reacts about 1-2 weeks later than agglutinationReacts about 1-2 weeks later than agglutination

11. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Characteristics of ELISA Detects primarily IgG, but also IgMDetects primarily IgG, but also IgM –ProFLOK ® Elisa’s are detecting light and heavy chain ! Highly sensitiveHighly sensitive Specificity of newer kits is quite goodSpecificity of newer kits is quite good –Expect about 5% of samples in suspect or low positive category in young breeders –These can be confirmed as negative by HI

12. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Reasons for Difficulties in Diagnosis Testing early after exposureTesting early after exposure –No antibodies MedicationMedication – Negative in culture, PCR… Mild strains of MG or MSMild strains of MG or MS –Weak immune response Strains which differ antigenicallyStrains which differ antigenically

13. Mycoplasma, Hungaria 18.03.2003 H.R. Froin SYNBIOTICS ProFLOK ® Mycoplasma Elisa’s Chicken MS and MG Chicken MS and MG Turkey MS, MG and MM Turkey MS, MG and MM USDA licensed USDA licensed Reliable, consistent results Reliable, consistent results Optimized to specifically work with chicken or turkey samples ( different cojugate) Optimized to specifically work with chicken or turkey samples ( different cojugate) 18 month shelf-life 18 month shelf-life Detects positive, negative and suspect titer levels Detects positive, negative and suspect titer levels High volume, easy-to-use format High volume, easy-to-use format

14. Mycoplasma, Hungaria 18.03.2003 H.R. Froin ProFLOK ® Specificity 1. Values are the arithmetic mean of 15 replicate samples (3 replicates/5 ELISA plates) 2 SP Ratio = OD Sample – Avg. OD NC Avg. OD PC – Avg. OD NC 3. Synbiotics Mg ELISA SP threshold ranges: Negative (-) < 0.199 Suspect (su) = 0.200 -0.599 Positive (+) > 0.600 4. Log10 titer = (1.464 x Log10 SP) + 3.197

15. Mycoplasma, Hungaria 18.03.2003 H.R. Froin ProFLOK ® Sensitivity 1. Values are the arithmetic mean of 6 replicate samples 3. Synbiotics Mg ELISA SP threshold ranges: Negative (-) < 0.199 Suspect (su) = 0.200 -0.599 Positive (+) > 0.600 4. Log10 titer = (1.464 x Log10 SP) + 3.197

16. Mycoplasma, Hungaria 18.03.2003 H.R. Froin ProFLOK ® Reproducibility A Values are the arithmetic mean ± SEM of one Mg positive chicken serum assayed 84 times per Mg ELISA plate. B Value is the mean % Coefficient of Variation (CV%) of one Mg positive chicken serum assayed 84 times per ELISA plate on each of ten Mg ELISA plates.

17. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Comparitive Study of Agglutination, HI and Elisa S. H. Kleven Department of Avian Medicine University of Georgia Data are from a lecture given during the IPE congress in Atlanta, USA in January 2003

18. Mycoplasma, Hungaria 18.03.2003 H.R. Froin An Easy Example: Birds were challenged by aerosol by MG, MS, or negative controls ELISA’s used: ProFLOCK® Mg or MS ElisaProFLOCK® Mg or MS Elisa –Using a suspect range Commercial Elisa’s (C –Kit)Commercial Elisa’s (C –Kit) –Only positive / negative !

19. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MS Serology / Controls

20. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MG serology / Controls

21. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MS Serology / MG Infected Birds were challenged by aerosol by MG

22. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MG Serology / MG Infected Birds were challenged by aerosol by MG

23. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MG Serology / MS Infected Birds were challenged by aerosol by MS

24. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MS Serology / MS Infected Birds were challenged by aerosol by MS

25. Mycoplasma, Hungaria 18.03.2003 H.R. Froin A Difficult Example: Birds were challenged by contact by MG, MS, or negative controls -> Spread of infection was slow and inconsistent

26. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MS Serology / Controls Control Birds

27. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MG Serology / Controls Control Birds

28. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MS Serology / MG Infected Birds were challenged by contact by MG

29. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MG Serology / MG Infected Birds were challenged by contact by MG

30. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MG Serology / MS Infected Birds were challenged by contact by MS

31. Mycoplasma, Hungaria 18.03.2003 H.R. Froin MS Serology / MS Infected Birds were challenged by contact by MS

32. Mycoplasma, Hungaria 18.03.2003 H.R. Froin Conclusions The ProFLOK ® MG and MS Elisa are showing: The ProFLOK ® MG and MS Elisa are showing: Superior sensitivity ( No false negative !) Superior sensitivity ( No false negative !) Excellent specificity Excellent specificity Better interpretation due to suspect range Better interpretation due to suspect range High reproducibility High reproducibility