Production and use of Artemisia annua (sweet wormwood) against bacterial diseases in poultry stocks and its effect on meat quality.

Transcription

1 Production and use of Artemisia annua (sweet wormwood) against bacterial diseases in poultry stocks and its effect on meat quality. X.C. Fretté, R.M. Engberg, A. Kjær, E. Ivarsen, K.B. Christensen, K. Grevsen, C. Bejerholm, M. Jensen and L.P. Christensen

2 Artemisia annua L. Systematic Angiosperm plant, Order: Asterales, Family: Asteraceae Common names Sweet wormwood, Annual wormwood, Quinghao Pharmacological properties Antimalarial, anticancer, antibacterial activities Photo : University of Florida Herbarium 2

3 Uses of Artemisia annua Antimalaria activity of Artemisia annua Malaria, a mosquito-borne infectious disease Caused by eukaryotic protist Plasmodium (eg. P. falciparum) Treatments against malaria ACT artemisinin combination therapy Artemisinin and derivatives Artemisinin (1) (sesquiterpene lactone with peroxide bridge) Artesunate Artemether Antibacterial activity of Artemisia annua Essential oil components Camphor, germacrene D, 1,8-cineole, -caryophyllene... 3

4 Background of the project Necrotic enteritis (NE) in broiler flocks Most common poultry disease Caused by toxin producing types of Clostridium perfringrens (Gram-positive anaerobic bacterium) Economic losses due to reduced productivity and high mortality Infection with coccidia (Eimeria spp.) predisposing factor for NE Treatment with antibiotics such as bacitracin, penicillin and lincomycin Before 2005 NE was controlled by antibiotics provided as feed additives (AGPs: Antibiotic growth promoters) After withdrawal of AGPs, NE is controlled by ionophore coccidiostats as feed additives as they have an effect against coccidia and C. perfringens 4

5 Hypotheses of the project Necrotic enteritis (NE) in broiler flocks caused by toxins produced by a Gram positive bacterium Clostridium perfringens Artemisia is known for antibacterial activity attributed to essential oil components Coccidial infection is a parasitic infection and a predisposing factor for NE caused by sporozoa (Eimeria spp.) in chicken, a unicellular parasite as Plasmodium falciparum Artemisia is known for antiparasitic activity attributed to artemisinin and derivatives Hence Artemisia annua could be used as an additive in poultry feed to prevent NE 5

6 Cultivation of Artemisia annua in Denmark Cultivation parameters investigated Plant variety Plant density Stress treatment Photo : K. Grevsen 6

7 Source : K. Grevsen Cultivation of Artemisia annua in Denmark Artemis cultivar from Médiplant (CH) with following specifications: 7

8 Yield (t/ha) Cultivation of Artemisia annua in Denmark Artemisia annua total FW an DW yield in plant density trial Source : K. Grevsen FW yield t/ha DM yield t/ha 50 a bc c ab c Plant density (plants m -2 ) 8

9 Cultivation of Artemisia annua in Denmark Source : A. Kjær Jasmonic acid is a naturally occuring growth regulator found in higher plants. It has also been proposed as a key regulator of plant responses to pathogens and insects. 9

10 In vitro antimicrobial activity of A. annua extracts against Clostridium perfringens 3 extracts tested on Clostridium perfringens strain isolated from diseased poultry flock Extraction of A. annua with hexane (Hex) Extraction of A. annua with dichloromethane (DCM) Extraction of A. annua with methanol (MeOH) Photo : X. Fretté Solvent extract MIC (ppm) Hexane (Hex) 170 Dichloromethane (DCM) 270 Methanol (MeOH) >700 Source : R.M. Engberg 10

11 In vitro antimicrobial Source : R.M. Engberg activity of hexane fractions of A. annua against C. perfringens Hexane extract chromatographed on silica phase giving 16 fractions. All fractions were tested for antibacterial activity against Clostridium perfringens Fractions from Hexane extract MIC (ppm) 1, 2, 5, 9-11, > 600 ppm 6-8,

12 Chemical composition of fractions 3 & 4 mau DAD1 B, Sig=280,16 Ref=450,100 (ELISE\091007\ \ D) 2000 Hexane extract min hexanbioassay #4 hexanbioassay 3 UV_VIS_1 mau WVL:220 nm 4-25,877 polyacetylene F , , , , min 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0 32,0 34,0 36,0 38,0 40,0 42,0 45, hexanbioassay #5 hexanbioassay 4 UV_VIS_1 mau WVL:220 nm 8-25, F , , , , , , , , , , , , , min ,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0 32,0 34,0 36,0 38,0 40,0 42,0 45,0 12

13 Feeding trial with broilers 320 broilers (Ross 308) 4 treatments (4 replicate pens à 20 broilers) Group 1: No supplementation Group 2: Hexane extract supplementation (125 ppm) Group 3: Hexane extract supplementation (250 ppm) Group 4: Hexane extract supplementation (500 ppm) foto: Artemisia annua (Hexane extract) 1-35 days Control 125 ppm 250 ppm 500 ppm SEM P-value Body weight (g) Feed intake (g) 3088 a 2948 a 2846 b 2644 b FCR (kg feed/kg gain) Conclusion: 250 ppm hexane extract of Artemisia annua did not affect weight gain of broilers but improved feed conversion ratio (FCR) 13

14 Sensoric analysis of broiler meat Sensory panel 8 persons Trained according to standards ISO and ASTM STP 758. Meat preparation Boiled 20 minutes Monitoring 18 attributes (4 odour, 5 flavour, 2 appearance, 7 texture) Statistical analysis with a mixed-design analysis of variance model 14

15 Source : C. Bejerholm Sensoric analysis of broiler meat Attributes Treat. 1 Control Treat ppm Treat ppm Treat ppm Std. error P-value Odour Boiled chicken Odour Poultry Odour Sweet Odour Sour Appearance Homogenous colour Appearance Colour Flavour Boiled chicken Flavour Acidic Flavour Bitter Flavour Metallic Flavour Sweet Texture Firmness Texture Moisture Texture Tenderness Texture Fray Texture Crumbling Texture Chew residual Texture Chew time % time preparation Conclusion: Dietary supplementation of Artemisia annua hexane extract up to 500 ppm did not affect breast meat quality of broilers 15

16 Necrotic enteritis infection trial 320 day old broilers (Ross 308) Experimental design 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: Artemisia annua dried plant material (1%), infection Group 4: Artemisia annua hexane extract (250 ppm), infection Monitoring Body weight 17, 24 and 27 days Small intestinal lesion scoring on 5 birds/pen on days 22, 24, 27 Clostridium perfringens counts in content of small intestine and ileum (pooled samples of 5 birds per replicate) 16

17 Source : R.M. Engberg Necrotic enteritis infection trial g Weight gain of the broilers in the period from day 17 to day 27 a c c b Control, non-infected Control, infected Dried plant (1%) n-hexane extract (250 mg/kg) Conclusion: Dietary supplementation of A. annua hexane extract improved weight gain compared to non treated infected control birds. 17

18 Lesion scoring Score 0 No gross lesions 1 Thin and friable walls 2 Focal necrosis or ulceration (1 to 5 foci) 3 Focal necrosis or ulceration (6 to 15 foci) 4 Focal necrosis or ulceration (16 or more foci) 5 Patches of necrosis (2-3 cm long) 6 Diffuse necrosis Scoring system for lesions associated with necrotic enteritis (Keyburn et al., 2006) 18

19 Small intestinal lesions in broilers with necrotic enteritis Thin and friable intestinal walls Focal necrosis and ulceration Focal necrosis and severe ulceration Diffuse necrosis 19

20 Source : R.M. Engberg Necrotic enteritis infection trial Small intestine lesion scores Control, noninfected Control, infected Dried plant (1%), infected n-hexane extract (250 ppm) infected SE P-value 22 days 0 a c 1.30 c 0.60 b 0.16 < days 0 a 3.65 b 2.75 b 2.65 b days 0 a 1.75 c 1.30 c 0.60 b 0.16 < Means in the same row with different superscripts differ significantly (P<0.05). Conclusion: Dietary supplementation of A. annua hexane extract significantly reduced small intestinal lesion scores in infected broilers. 20

21 Source : R.M. Engberg Necrotic enteritis infection trial Numbers of Clostridium perfringens in the contents of caeca (log CFU/ g) Control, noninfected Control, infected Dried plant (1%), infected n-hexane extract (250 ppm) infected SE P-value 22 days 3.27 c 8.31 a 7.92 ab 7.10 b 0.34 < days 5.45 b 8.53 a 8.30 a 7.90 a days 2.31 c 6.83 a 7.23 a 6.09 b 0.16 < Means in the same row with different superscripts differ significantly (P<0.05). Conclusion: Dietary supplementation of A. annua hexane extract significantly reduced caecal numbers of Clostridium perfringens. 21

22 Conclusion These results indicate that the dietary addition of A. annua hexane extract had a positive influence on the course of the disease in terms of a later disease onset and a faster recovery of the birds. Artemisia annua could therefore be considered as a promising substitute to ionophore dietary additives in poultry production without impact on meat quality. 22

23 Acknowledgements to. For project funding..for sensoric analysis..for cultivation of Artemisia annua and animal experiments..for extraction and fractionation of A. annua and chemical analyses 23

24 Thank you for your attention Teşekkür ederim Do you have questions? 24