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

Artemisia annua

• Common names– Sweet wormwood, Quinghao

• Pharmacological properties– Antimalarial, antibacterial,

anticancer

• Antimalarial properties• Artemisinin and derivatives• Artemisinin, Artesunate, Artemether

• Antibacterial properties• Essential oil components• Camphor, germacrene D, 1,8-cineole, -

caryophyllene….

Hypothesis of the project

Necrotic enteritis (NE)• Bacterial exotoxins produced by

Clostridium perfringens type A (Gram positive anaerobic bacterium)

• Coccidial infection as a predisposing factor for NE, caused by Eimeria spp., unicellular parasites, belongingto Sporozoa like Plasmodium falciparum (malaria)

Artemisia annua• Antibacterial activity

attributed to essential oil components

• Antiparasitic activityattributed to Artemisinin and derivates

Artemisia annua could possibly be used as feed additive to prevent NE in poultry

In vitro antimicrobial activity of Artemisia annua extracts towards

Clostridium perfringens

• 3 extracts tested on Clostridium perfringens strain isolated from diseased poultry flock• Extraction of A. annua with hexane• Extraction of A. annua with dichloromethane• Extraction of A. annua with methanol

Solvent extract MIC (ppm)

Hexane 170

Dichloromethane 270

Methanol > 600

Experimental design

• 320 day old broilers (Ross 308)• 4 treatments (4 replicate pens à 20 broilers)

• Group 1: No Artemisia annua supplementation, no infection• Group 2: No Artemisia annua supplementation, infection• Group 3: With Artemisia annua dried plant material (10 g/kg), infection)• Group 4: With Artemisia annua hexan 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 perfringens counts in caecal content

(pooled samples of 5 birds per replicate)

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).

Scoring of small intestinal lesions

Thin and friable intestinal walls

Score

0 No gross lesions

1 Thin and friable wall

2 Focal necrosis or ulceration (1 to 5 foci)

3 Focal necrosis or ulceration (6 to15 foci)

4 Focal necrosis or ulceration (16 or more foci)

5 Patches of necrosis (2-3 cm long)

6 Diffuse necrosis

Focal necrosis or ulceration

Focal necrosis or ulceration

Diffuse necrosisScoring system for (Keyburn et al., 2006)

Small intestinal lesion scores and Clostridium perfringens numbers in caecal content

ControlNon-

infected

Control infected

Dried plant 10 g/kg

infected

n-hexane 250 mg/kg

infected

Significance

Lesion score

22 days 0 c1 1.75 a 1.30 a 0.60 b ***

24 days 0b 3.65 a 2.75 a 2.65 a ***27 days 0 c 1.75 a 1.30 a 0.60 b ***C. perfringens(log cfu/g)22 days 3.27 c 8.31 a 7.92 ab 7.10 b ***24 days 5.45 c 8.53 a 8.30 a 7.90 a **27 days 2.31 c 6.83 a 7.23 a 6.09 b ***

1 Means in the same row with different superscripts differ significantly (P<0.05)

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

Control, non-infected Control, infected Dried plant (10 mg/kg ) n-Hexane extract (250 mg/kg)0

100

200

300

400

500

600

700g a bcc

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 annua modulates the course of the disease in terms of a later disease onset and an earlier recovery.

• The n-hexane extract of Artemisia annua can to a certain extent prevent severe growth depression related to the disease.