2. "Clinical Trials and Clinical Endpoints" James R. Bradford, DVM, Dipl. ABVP Pharmacia Animal Health

3. Mycoplasmal Pneumonia  Clinical and production disease  Recognized financial impact early ’70’s  Research with Lincomycin began in 1974  FDA approval “for the reduction in severity of pneumonia due to Mycoplasma hyopneumoniae” granted in 1978

4. Mycoplasmal Pneumonia of Swine Is…  A distinct clinical disease with distinct lesions and a recognized cause  Often part of a more severe respiratory disease complex Potentiator for Pasteurella multocida, some APP, PRRSV (Pijoan, Thacker) Additive with Swine Influenza Virus (Thacker and Halbur)  Treatable as a single entity or as part of a complex

5. Clinical Endpoints  % of pigs affected  Number of lobes involved in each pig  % of lung involved  Respiratory score Cough Respiratory difficulty

6. Pivotal Studies  In vitro MIC determination for challenge strain correlated with concentration at site of infection  Challenge trials - dose titration, duration of treatment  Field efficacy trials – dose confirmation, fixed duration

7. MIC Determination  In vitro MIC and susceptibility testing Strain 11 MIC 0.2 µg/mL  Pigs fed lincomycin at 110 ppm have a mean lung concentration of 0.6 µg/g (Range 0.40-0.71)

8. Challenge Model  Must have pigs free of M. hyopneumoniae  Usually very mild disease compared to field challenge  All animals at same stage of infection  Can treat at earliest appearance of clinical signs  May give false sense of efficacy

9. Challenge Trial – Materials and Methods  Single barn  M. hyopneumoniae-free pigs 4-6 weeks of age  Randomly allotted to treatments based on sex, weight, and age  Challenged pigs and unchallenged controls housed in same barn in separate air spaces (confirmation of negative status)

10. Challenge Method  All pigs in challenged groups Challenged 2 consecutive days 2 doses each day (intranasal and intratracheal) Fresh lung homogenate in Eagle’s medium  Non-challenged controls were inoculated with Eagle’s medium

11. Treatments – Challenged Pigs Duration Inclusion rate 7 days14 days21 days 0 ppm3 pens 110 ppm3 pens 220 ppm3 pens 330 ppm3 pens

12. Unchallenged Pigs  Separated by solid partitions  Confirm that M. hyo challenge caused the lung lesions  4 pens – 3 non-medicated - validate M. hyo status one medicated – demonstrate swine dysentery control or growth effect was not responsible for performance benefits

13. Treatment Initiation  At onset of clinical signs (Clinical disease present)  Expected to be day 10 PI  Actually day 8 PI – 13.6% of pigs coughing

14. Clinical Endpoints  % of lobes with lesions – incidence measure  % of total lung with lesions – severity measure

15. Lesions Observed a a b b aa b b a,b different, p<.05

16. Summary Lesion Data % lobes % total lung 0 ppm74.611.6 110 ppm7512.4 220 ppm464.4 300 ppm554.9

17. Duration of Treatment % Lobes with Lesions-Pooled Data

18. Duration of Treatment % Lung Involved-Pooled Data

19. Duration of Treatment % Lobes with Lesions

20. Duration of Treatment % Lung with Lesions

21. Performance Parameters a a bb a a b b a,b different, p<.05

22. Conclusion  In the challenge model, lincomycin at 220 and 330 ppm resulted in a significant reduction in severity and incidence of lesions caused by M. hyopneumoniae.  The optimum treatment regime based on lesion and performance data was 220 ppm for 21 days.

23. Field Trials  Herds with history of problems with MPS  Pigs at an age already likely infected with MPS

24. 4 Field Trials  Indiana, Florida, Alabama, and Minnesota  Pigs 8-12 weeks of age  6 cohort pigs killed and necropsied to confirm disease  3 sites tested 4 inclusion rates: 0,110, 220, 330 ppm  1 site tested 0, 220 ppm only

25. Clinical Observations  Gross Pathology Number of lobes involved Estimated percent of total lung involved  Microscopic Pathology Right cardiac lobe sampled based on lesion location for histopathologic scoring  Production Parameters (ADG and ADFI)

26. Results  Data from all 4 sites was pooled, making the necessary statistical adjustments to compensate for unequal numbers of replications, pens and pigs at each site.

27. Replications per Treatment Inclusion rateReplications 015 11012 22015 33012

28. % Pigs with MPS Lesions An estimate of the number of pigs exposed for sufficient time to develop lesions

29. % Lobes Affected –All Pigs Lincomycin suppressed the advancement of the disease process. % a ab b b a,b different, p<.05

30. % Lobes Affected – Pigs With Lesions Lesions may or may not have been present prior to treatment. %

31. Mean % Total Lung Involved All Pigs 220 ppm reduced severity by 49%, 110 ppm by 27%(ns) a ab b b a,b different, p<.10 %

32. Mean % Total Lung Involved Pigs With Lesions %

33. ADG (kg)– Least square Means a b b b K g/ da y a,b different, p<.05

34. Feed /Gain a,b different, p<.05 a ab bb

35. Lesions Scored Microscopically for Aging of MP Lesion AcuteSubacute regressing Chronic resolved Other Pneumonias earlylate 049 a 560a0a 10 11022 b 191714 ab 6 2205c5c 81229 bc 12 3300c0c 42238 c 6 a,b,c Means in the same column with different superscripts differ significantly (p<.05) % of Pigs

36. Trial Conclusion  Lincomycin administered in the feed at 220 ppm for 21 days is effective in the reduction of pneumonia lesions associated with Mycoplasma hyopneumoniae.

37. Conclusions Based on 25 Years Experience  It is possible to measure clinical endpoints of mycoplasmal pneumonia both in clinical trials and in field trials.  The approval process to provide substantial proof of efficacy was was rigorous and has stood the test of time.  Production parameters are an important auxiliary component of the research process, but not the approval process.

38. If We Designed a Plan Today…..  Similar approach Laboratory – in vitro determination of efficacy correlated with drug at the site of infection Challenge – validated challenge models run in production-like facilities for dose and duration selection Field trials – field efficacy trials with dose and duration confirmation

40. If We Designed a Plan Today…..  Field efficacy trials – Multi-location – production units History of respiratory disease with M. hyopneumoniae as a key component Co-infections with PRRSv acceptable/welcome Experimental unit ? – pen, room, barn, site

41. If We Designed a Plan Today…..  Clinical Endpoints % lung involved and % pigs affected would still be critical measurements % lung involved could be measured more accurately today with grids and computer calculation and could be measured on a sample of pigs killed over the course of the trial Fluorescent antibody test would provide better confirmation of cause of pneumonia

43. Lincomix effects on culling Pharmacia - Bradford 9/00

44. Lincomix pulled