Echinacea purpurea as a potential immunostimulatory feed additive ...

Livestock Science 122 (2009) 81–85

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Echinacea purpurea as a potential immunostimulatory feed additive in laying hens and fattening pigs by intermittent application Barbara M. Böhmer a,⁎, H. Salisch b, Brigitte R. Paulicks a, F.X. Roth a a b

Technical University Munich, Hochfeldweg 6, D85350 Freising-Weihenstephan, Germany Bavarian Animal Health Service, Senator-Gerauer-Str. 23, D85586 Poing, Germany

a r t i c l e

i n f o

Article history: Received 6 March 2008 Received in revised form 3 July 2008 Accepted 5 July 2008 Keywords: Echinacea purpurea Immune parameter Performance Laying hens Fattening pigs

a b s t r a c t The aim of the present trial was to evaluate the effect of repeated short-time applications of Echinacea purpurea juice as feed additive to initiate an immune response. In a trial with 70 white layers (LSL) two Echinacea juices as feed additives were tested for their influence on feed intake, performance, blood cell counts, phagocytosis of granulocytes, and antibody titer against Newcastle Disease Virus (NDV) following vaccination. Echinacea juice was pressed juice of aerial parts of the plant preserved with either ethanol (20%) or by fermentation. The application scheme followed a repeated 14 days regimen with either 5 days of application and 9 days without or 2 days of application and 12 days without. Blood was taken several times following an application period. The performance of the hens was not impacted. Significant changes were found in the number of lymphocytes (p b 0.002), phagocytosis rate (p b 0.02), and NDV antibody titer (p b 0.01). The number of lymphocytes was highest in the group receiving ethanolic juice for five consecutive days. Phagocytosis was reduced in both groups provided with ethanolic juice (2 or 5 days). Highest NDV antibody titers were seen in the groups receiving fermented juice for 2 days. Additionally, phagocytosis of granulocytes was determined in fattening pigs (80 – 100 kg) after 5 days of Echinacea application with ethanolic or fermented juice. A significant increase (p b 0.005) was found with both Echinacea formulations. The number of lymphocytes was also increased significantly in the groups provided with Echinacea (p b 0.008). © 2008 Elsevier B.V. All rights reserved.

1. Introduction Echinacea purpurea is one of the most important medical herbs. It is widely used around the world to treat common cold and other infectious disorders with the claim to have paramunity-inducing and non-specific immune responses stimulating effects. The main constituents present in juices of Echinacea with reported bioactivity are caffeic acid and alkamids, although, the precise active principle is still unknown (Bauer and Wagner, 1991). A recent review shows that Echinacea treatment results in an increase of various

⁎ Corresponding author. Lehrstuhl für Tierernährung, TU München, Hochfeldweg 6, D85354 Freising-Weihenstephan, Germany. Tel.: +49 8161 713116; fax: +49 8161 715367. E-mail address: [email protected] (B.M. Böhmer). 1871-1413/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2008.07.013

cytokines, lymphocytes, and phagocytosis activity (Bauer, 2002; Sasagawa et al., 2006). The most commonly used preparation is ethanolic juice, and the recommended application is prophylactic daily use for several weeks (BGA Commission E, 1989). In contrast to this recommendation it was shown by Jurcic et al. (1989) that phagocytosis rate of granulocytes in human blood was increased during the first 5 days of application and depressed afterwards as a result of over-stimulation. Studies with Echinacea to improve animal husbandry are rare. Kuhn et al. (2005) reported an immune stimulating effect in sows by a repeated 5 days of application of Echinacea juice. In contrast to their study Maass et al. (2005) did not find significant effects during a continuous application. The objective of the present study was to test a repeated short-time application regime for Echinacea juice as feed additive on phagocytosis, antibody response, and blood cell counts of layers and fattening pigs.

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B.M. Böhmer et al. / Livestock Science 122 (2009) 81–85

2. Materials and methods

Table 2 Test protocols for the hemagglutination–inhibition tests for antibody titers

2.1. Laying hens 70 white layers (LSL, 32 – 54 weeks of age) were randomly assigned to 5 groups with each 14 birds and housed individually in cages. The hens had received a live attenuated vaccine against IBV during grower phase at the breeder. Vaccination against NDV (live attenuated vaccine) was repeated every 8 weeks. The diet was based on corn, wheat, and soybean meal providing 11.5 MJ ME/kg and 172 g crude protein/kg feed to meet or exceed the German feeding standards for poultry (GfE, 1999). The trial comprised twelve 2-weeks periods. Each period was divided into 2 or 5 days of application of Echinacea juice followed by 12 or 9 days without application, respectively (Table 1). The dosage of the Echinacea juice for hens was adjusted on the basis of human medical recommendations to 0.25 ml/kg BW0.75 due to a lack of data for poultry. E. purpurea was applied in the form of pressed juices from the aerial parts of the plant. The juices were produced in a patented process (Berghof Kräuter GmbH, Heilsbronn, Germany), where pressing and mixing were done immediately after the cutting of the plant in a special harvester. Ethanolic juice consisted of 80% (w/ w) native juice and 20% (w/w) ethanol as preservative (caffeic acid 2.1 mg/ml, alkamids 27.7 µg/ml). Fermented juice was native pressed juice after fermentation for preservation (caffeic acid 1.9 mg/ml, alkamids 2.7 µg/ml). The juices were standardized at a level of 6.5% DM. 2.2. Experimental parameters In layers the experimental parameters were feed intake (weekly), feed conversion (kg feed/ kg egg mass), daily egg weight, daily egg mass, laying performance, differential blood count, phagocytosis rate of granulocytes, and some parameters related to humoral immunity (antibody titers regarding Newcastle Disease [ND], Egg Drop Syndrome [EDS], and Infectious Bronchitis [IB]). Blood was taken from the vena ulnaris 4 times following an application phase. Antibody titers against ND were tested one and two weeks after vaccination (Nobilis ND Hitchner, Intervet Deutschland GmbH), respectively. Antibody titers against EDS was tested without preceding vaccination. 2.3. Analytical procedures The contents of cichory acid and alkamids in the Echinacea juices were analyzed in the Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Austria, using a HPLC method (Bauer,1997). Differential blood counts of avian lithium heparin blood were done at the Bavarian Animal Health Service using an Abbott Cell-Dyn 3500 (Abbott GmbH Diagnostika,

Table 1 Experimental design for layers Group

1 2

3

4

5

Juice Days of application Days without application Duration of one period Number of periods

– – – – –

Fermented 5 9 14 12

Ethanolic 2 12 14 12

Fermented 2 12 14 12

Ethanolic 5 9 14 12

Testplates PBS — 1st well Serum — inactivation Serum — 1st well Serum — dilution Antigen — source HA—units Antigen — volume Pos. control Incubation Erythrocytes — volume Incubation

EDS

NDV

IBV

u-shaped 25 µl 56 °C, 30 min. 25 µl log2 — 25 µl GD, Deventer 4 25 µl GD, Deventer 10 min., 25 °C 50 µl 45 min, 25 °C

u-shaped 25 µl 56 °C, 30 min. 25 µl log2 — 25 µl GD, Deventer 4 25 µl GD, Deventer 20 min, 25 °C 50 µl 45 min, 25 °C

v-shaped 25 µl 56 °C, 30 min. 25 µl log2 — 25 µl GD, Deventer 4 25 µl GD, Deventer 20 min, 2–8 °C 25 µl 45 min, 2–8 °C

Wiesbaden, Germany) automatic cell counter. Counts were done by following manufacturers instructions and using original chemicals. Hemagglutination–inhibition (HI) tests for antibody titers (ND; EDS, IB) were basically performed according to Table 2. Erythrocytes in Alsever's solution (Fiebig, Idstein-Niederaudorf, Germany) were washed in phosphate buffered saline (PBS) until the supernatant was clear. Erythrocytes were diluted to 1% in phosphate buffer (45 g NaCl, 2 g KH2PO4, 7 g Na2HPO4 ad 5000 ml Aqua bidest., pH was adjusted to 7.2–7.4). In order to exclude unspecific agglutination parallel testing without HA-antigen was performed. Additionally, HAantigen was back titrated. Generally testing was performed in duplicate. Phagocytosis rate of the granulocytes was determined by measuring zymosan-induced and luminol amplified chemiluminescence according to Kato et al. (1981) using fresh heparinized blood. The chemiluminescence response was monitored for 40 minutes using a microplate luminometer (Luminoskan Ascent®, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Counts per minute (cpm) were plotted simultaneously. The maximum value of each sample was used and expressed relative to 103 granulocytes as a comparable value (cpm 10− 3/103 granulocytes). 2.4. Fattening pigs In a complementary trial 36 fattening pigs with a body weight ranging from 80 to 100 kg were randomly assigned to 3 groups with 12 animals each. The diet was based on barley, corn, wheat, and soybean meal providing 13.2 MJ ME/kg and 143 g crude protein/kg feed to meet or exceed the German standards for pigs (GfE, 2006). The trial comprised 19 days divided into 5 days with application of Echinacea, 9 days without application, and again 5 days with application. The dosage of the Echinacea juice was adjusted on basis of recommendations for humans at 2.5 ml/kg feed. Group 2 was given the ethanolic formulation of juice while group 3 was supplemented with fermented juice. Group 1 served as a non-treated control. Blood was taken from the jugular vein on the last day of the trial for the differential blood counts and determination of rate of phagocytosis. 2.5. Statistical evaluation Data were tested with Kolmogorow–Smirnow-test for normal distribution and subjected to two-way-analysis of variance with the factors applications days and type of juice


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Table 3 Effects of Echinacea feed additives on immune parameters of laying hens and fattening pigs Group Type of juice

1 –

2 Ethanolic

3 Fermented

4 Ethanolic

5 Fermented

Application days

0

5

5

2

2

4799 ± 3641 324 ± 655 56 ± 114 2021 ± 2907 12165 ± 3330b 18793 ± 5997ab

4656 ± 2051 355 ± 231 44 ± 89 1848 ± 1141 14639 ± 3690a 21543 ± 5267a

2995 ± 1428 268 ± 207 13 ± 47 1527 ± 922 12320 ± 2320b 17123 ± 2988b

4068 ± 1951 244 ± 152 52 ± 113 1348 ± 921 10717 ± 1599b4 16429 ± 2941b

4325 ± 2280 399 ± 292 30 ± 75 2584 ± 4704 10435 ± 2577b4 16884 ± 4446b

428c 488b 398b 283d

525a 465bc 368bc 256e

520a 476bc 374b 325c

486b 454c 344c 434a

527a 533a 428a 391b

0.42 ± 0.17a

0.26 ± 0.08c5

0.37 ± 0.16abc

0.27 ± 0.11bc5

0.39 ± 0.16ab

2951 ± 855 488 ± 193 106 ± 26 1234 ± 400 12570 ± 2908b 17133 ± 3491b

3100 ± 604 566 ± 158 155 ± 62 1275 ± 423 15275 ± 3654a 20275 ± 3592a

3024 ± 710 559 ± 147 111 ± 49 930 ± 282 11218 ± 2078b 16167 ± 3307b

1.5 ± 0.4b

2.1 ± 0.5a

2.1 ± 0.6a

– – – – – – – –

– – – – – – – –

Layers Differential blood count (abs./µl)2 Heterophils Eosinophils Basophils Monocytes Lymphocytes Total leucocytes Antibody count3 Titer ND Titer ND Titer EDS Titer IB Phagozytosis2 (10− 3cpm/103 Granul.) Luminescence Fattening pigs Differential blood count (abs./µl)1 Neutrophils Eosinophils Basophils Monocytes Lymphocytes Total leucocytes Phagozytosis1 (10− 3 cmp/103 Granul.) Luminescence a,b

Significant differences are marked with different superscript. Significant differences are marked with different superscript: Student–Newman–Keuls-test, p b 0.05. Significant differences are marked with different superscript: Duncan-test, p b 0.05. 3 According to Wilcoxon and Wilcox-test. 4 Significance of factor application days (p b 0.005). 5 Significance of factor type of juice (p b 0.005). 1 2

by using SAS statistical package (version 6.08, 1989). Significant effects of dietary treatments were compared by using Student–Newman–Keuls-test (SNK) and Duncan's multiple range test. HI-titers were compared using a multiple Wilcoxon and Wilcox-test. In the following tables, data are shown as means ± standard deviation. Values with different superscript differ significantly (p b 0.05) between treatments.

compared with the other groups. Phagozytosis rate of granulocytes was decreased in all experimental groups when compared with results obtained for the control hens. A significant decrease of phagocytosis rates was found in groups 2 and 4, which were supplemented with ethanolic juice (p b 0.02). Here the factor type of juice was significant (p b 0.005).

3. Results

3.2. Fattening pigs

3.1. Laying hens

The number of lymphocytes and the total number of leucocytes (Table 3) revealed significant differences. Group 2 had higher values compared with group 3 and the control group. Phagozytosis rate showed significantly higher values in the experimental groups 2 and 3 compared with the control group (p b 0.005).

The performance of the hens was not affected by Echinacea treatment. Regarding the differential blood counts (Table 3), total numbers of leucocytes (p b 0.02) and lymphocytes (p b 0.02) were highest in group 2. Lymphocytes showed a significant influence of the factor application days (p b 0.001). Significant differences were determined for ND-, EDS-, and IB-antibody titers. ND antibody titers one week after vaccination were significantly higher in all experimental groups when compared with control group 1 with the highest levels in group 5 (p b 0.01). The ND titers two weeks after vaccination showed better uniformity. Titers of the control group were similar to groups 2 and 3 while group 5 again showed highest antibody levels. The EDS antibody titers were similar in groups 1 to 3, while group 5 had the highest antibody levels and group 4 was lower than the control group. The titer of IB was significantly higher in groups 4 and 5 when

4. Discussion The application of Echinacea juice should booster immunological reactivity and should contribute to better health and improved performance. In the present trials, hens and pigs revealed a common trend. Number of lymphocytes and total leucocytes were significantly higher (p b 0.05) in groups receiving ethanolic juice for 5 days. This effect was seen independently in both species and gives a hint on a specific influence of the ethanolic juice when administered for several consecutive days. This is in agreement with Cundell et al.

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(2003), who found a significant increase of lymphocytes after one week in rats fed with dried Echinacea preparations. Probably the ethanolic variant has besides a higher concentration of alkamids a different ratio of components causing this effect. It was shown in a previous trial that ethanol by itself has no effect, because of its volatilization during processing and storage of the feed (Maass et al., 2005). Alkamids in Echinacea are discussed by Woelkart and Bauer (2007) to be considered as a class of cannabinomimetics as a possible mode of action to modulate immune functions. Increase of phagocytic activity is a characteristic reaction associated with efficacy of Echinacea (Heidl, 1991; Barrett, 2003). Allen (2003) found a stimulation of the phagocytic response in chickens provided with ground root preparations. Bauer et al. (1988) reported that phagocytosis in mice was enhanced after treatment with ethanolic extracts of Echinacea. In the present trial, the phagocytosis rate of granulocytes was significantly decreased in hens and significantly increased in fattening pigs. Laying hens showed a decrease of activity when administered with ethanolic juice, which could be a consequence of the different alkamid concentrations of the juices. Mainly alkamids are responsible for the increase of the phagocytic activity (Goel et al., 2002; Sasagawa et al., 2006). The role of alkamids was recently upgraded by Woelkart and Bauer (2007) as having antiinflammatory properties similar to anandamids and canabinoids. Additionally, the dosage of Echinacea has an important influence. Gaisbauer et al. (1990) could prove in human whole blood that only a low dose of Echinacea could enhance phagocytosis. A higher dosage reduced the number of granulocytes with phagocytic activity. The reduction of phagocytosis in the present layer trial could result from an overdose of alkamids in the ethanolic juice preparations. Probably, it could be necessary to adjust the dosage for layers by considering the alkamid content of crude juices and not using dry mass as the basic value for adjustment of Echinacea concentration. Regarding antibody titers of the layers a significant effect of the Echinacea supplementation was evident. The ND antibody titer one week after vaccination revealed that there is a rapid and strong setting of the response in the groups treated with Echinacea, which is important for reliable protection. The treatment with fermented juice for 2 days (group 5) resulted in the highest values. The concurrent infections with EDS and IB were naturally occurring diseases seen under field conditions as well. Here, the highest values for the antibody titer were found also in the 2-day treatment with fermented juice. The 5-day treatment, especially with ethanolic juice, seemed to have a more adverse effect by lowering the immune response. Unfortunately, it is not quite clear if the fermented juice has actually more potential than the ethanolic juice or if it is only an effect of the lower alkamid content or of another composition of the fermented juice. Zhai et al. (2007) also found an increased antibody response (B-cells) in mice administered with alcohol extracts of Echinacea. So far, there are no studies available on the possible mode of action of activating the specific immune systems by plant components. It is possible that the presence of pathogen associated molecular patterns, which are recognized by toll-like receptors and other pattern recognition receptors, can contribute to affect the specific immune system. This potential of Echinacea to stimulate adaptive immune functions can be helpful in practical animal husbandry to enhance the effects of vaccinations.

The accidental infection with EDS and IB was diagnosed by increased antibody titer in screening routine, which is a commonly used indicator for virus contact. There was no drop of laying performance or reduction of viability of the hens. This mild course of infection might reflect an early infection of the hens before start of experiment or could be a beneficial effect of the prophylactic Echinacea application. This is similar to Schoop et al. (2006) who revealed in a meta-analysis with humans, that there was a shorter duration and fewer participants were suffering from a virus exposition (common cold) in humans administered with Echinacea. Alkamids in Echinacea preparations seemed to have additional mechanisms such as antiinflammatory and antiviral effects by reducing NO, TNF-α, various interleukins, and other parameters (Woelkart and Bauer, 2007), besides the effect of reducing the activity of bacterial hyaluronidases (Mutschler et al., 2008). 5. Conclusion In this study it was demonstrated, that a repeated shorttime application of Echinacea juice has immune stimulating effects in layers and fattening pigs. It seems that the repeated 2 days-trickle stimulation is sufficient to increase immune response. In this application regime, it is an easy and economic method to enhance immune reactions in layers and fattening pigs. A different dosage adjustment and application regime for layers in further studies will likely optimize the benefit from using Echinacea as feed additive. Acknowledgements The authors appreciate the analysis of the Echinacea juices by Prof. Bauer, Karl-Franzens-University Graz, Austria. The Echinacea juice was kindly provided by Berghof Kräuter GmbH, Heilsbronn, Germany. This study was supported by BAT e.V. (Bayerische Arbeitsgemeinschaft Tierernährung), Freising. References Allen, P.C., 2003. Dietary supplementation with Echinacea and development of immunity to challenge infection with coccidia. Parasitol. Res. 91, 74–78. Barrett, B., 2003. Medicinal properties of Echinacea: a critical review. Phytomedicine 10, 66–86. Bauer, R., 1997. Standardisierung von Echinacea purpurea-Presssaft auf Cichoriensäure und Alkamide. Z. Phytother. 18, 270–276. Bauer, R., 2002. Neue Erkenntnisse zur Wirkung und Wirksamkeit von Echinacea purpurea-Preßsaftzubereitungen. Wien Med. Wochenschr. 15 (16), 407–411. Bauer, R., Wagner, H., 1991. Echinacea species as potential immunostimulatory drugs. In: Wagner, H., Farnsworth, N.R. (Eds.), Economic and Medicinal Plant Research, vol. 5. London Academic Press Limited, pp. 253–321. Bauer, R., Jurcic, K., Puhlmann, J., Wagner, H., 1988. Immunologische in-vivound in-vitro-Untersuchungen mit Echinacea-Extrakten. Arzneim.Forsch./Drug Res. 38 (I), 276–281. BGA/BfArM (Kommission E), 1989. Bundesanzeiger 43 ATC-Code:L03AL. Cundell, D.R., Matrone, M.A., Rarjaczak, P., Pierce, J.D., 2003. The effect of aerial parts of Echinacea on the circulating white cell levels and selected immune function of the aging male Spague–Dawley rat. Intern. Immunopharmacol. 3, 1041–1048. Gaisbauer, M., Schleich, T.h., Stickl, H.A., Wilczek, I., 1990. Untersuchung zum Einfluß von Echinacea purpurea Moench auf die Phagozytose von Granulozyten mittels Messung der Chemielumineszenz. Arzneim.Forsch./Drug Res. 40 (I), 5, 594–598. GfE (Gesellschaft für Ernährungsphysiologie, Ausschuss für Bedarfsnormen), 1999. Empfehlungen zur Energie- und Nährstoffversorgung der Legehennen und Masthühner (Broiler). Nr. 7. DLG-Verlag, Frankfurt/Main.

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