The use of Artemisia annua in the prevention of necrotic enteritis in a broiler disease model

1. The use of Artemisia annua in the prevention of necrotic enteritis in a broiler disease model R.M. Engberg, K. Grevsen, E. Ivarsen, X. C. Fretté, L.P. Christensen

2. Artemisia annua • Commonnames • Sweetwormwood, Quinghao • Pharmacologicalproperties • Antimalarial, antibacterial,anticancer • Antimalarialproperties • Artemisinin and derivatives • Artemisinin, Artesunate, Artemether • Antibacterialproperties • Essentialoilcomponents • Camphor, germacrene D, 1,8-cineole, -caryophyllene….

3. Hypothesis of the project Necroticenteritis (NE) Artemisia annua Antibacterialactivityattributed to essentialoilcomponents Antiparasiticactivityattributed to Artemisinin and derivates • Bacterialexotoxinsproduced by Clostridiumperfringenstype A (Gram positive anaerobicbacterium) • Coccidialinfection as a predisposing factor for NE, caused by Eimeriaspp., unicellularparasites, belongingto SporozoalikePlasmodiumfalciparum (malaria) Artemisia annuacouldpossiblybeused as feedadditive to prevent NE in poultry

4. In vitroantimicrobialactivity ofArtemisia annua extractstowardsClostridiumperfringens • 3 extracts tested on Clostridium perfringensstrain isolated from diseased poultry flock • Extraction of A. annua with hexane • Extraction of A. annua with dichloromethane • Extraction of A. annua with methanol

5. Experimental design • 320 day old broilers (Ross 308) • 4 treatments (4 replicate pens à 20 broilers) • Group 1: No Artemisiaannuasupplementation, no infection • Group 2: No Artemisiaannuasupplementation, infection • Group 3: WithArtemisia annuadried plant material (10 g/kg), infection) • Group 4: With Artemisia annuahexan extract (250 mg /kg ), infection • Monitoring • Body weight 18, 23 and 26 days • Lesion scoring on 5 birds/pen on days 22, 24, 27 • Clostridium perfringenscounts in caecal content (pooled samples of 5 birds per replicate)

6. Disease model • Sudden feed shift to a diet providing 30% fish meal at the expense of soya meal on days 17, 18, 19 and 20. • A 10 fold overdose of an attenuated live vaccine against coccidiosis (Paracox 5 ®) on day 18. • Overnight culture of Clostridium perfringens(strain 48) mixed in the feed (107/g feed) on days 17, 18, 19 and 20. • A single dose of an overnight culture (strain 48) orally on day 21 (108/bird).

7. Scoring of small intestinal lesions Thin and friable intestinal walls Focal necrosis or ulceration Focal necrosis or ulceration • Scoring system for (Keyburn et al., 2006) Diffuse necrosis

8. Small intestinal lesion scores andClostridium perfringensnumbers in caecal content 1 Means in the same row with different superscripts differ significantly (P<0.05)

9. Body weight gain through the infection period (day 18 - day 27)

10. Conclusion • Using the present disease model, none of the feed additives can prevent small intestinal lesions related to necrotic enteritis. • The n-hexane extract of Artemisia annuamodulates the course of the disease in terms of a later disease onset and an earlier recovery. • The n-hexane extract of Artemisia annuacan to a certain extent prevent severe growth depression related to the disease.