- Email: [email protected]
Efficacy of allicin from garlic against Ascaridia galli infection in chickens
Efficacy of allicin from garlic against Ascaridia galli infection in chickens F. C. Velkers,*1 K. Dieho,* F. W. M. Pecher,* J. C. M. Vernooij,* J. H. H. van Eck,* and W. J. M. Landman*† *Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, the Netherlands; and †Animal Health Service (GD), Arnsbergstraat 7, 7418 EZ Deventer, the Netherlands ABSTRACT The use of garlic as a treatment against helminth infections is increasing in organic layer farms in several European countries. Its efficacy against these parasites, however, has not been demonstrated thus far. Therefore, a study was conducted to determine the efficacy of a commercially available garlic product consisting of a high concentration of allicin (i.e., the main active component of garlic) against experimentally induced Ascaridia galli infection in chickens. In total, 450 Lohmann LSL-Classic cockerels were used. Group 1, the uninfected, untreated group, consisted of 50 chickens. Groups 2 to 5, each consisting of approximately 100 chickens, were inoculated with 300 embryonated A. galli eggs/chicken at 6 wk of age. Group 2 was not treated, whereas groups 3 through 5 were given daily individual oral treatments from 13 wk of age onward.
Group 3 received the recommended dose of allicin for 2 wk, whereas group 4 received a 10-fold dose of allicin. Group 5 was given 10 mg of flubendazole/kg of BW for 1 wk. Necropsy of 20 birds of all groups was performed weekly between 13 and 16 wk of age to determine adult worm loads. Group 1 remained free of A. galli. The experimental infection in the other groups resulted in a mean adult worm load of approximately 16 worms/ bird. No significant differences were observed in worm counts of the allicin-treated groups (groups 3 and 4) compared with the infected, untreated group (group 2) at any week (P > 0.05). In contrast, no worms were found in chickens after flubendazole treatment (group 5). It was concluded that allicin does not represent an alternative to flubendazole for the treatment of A. galli infections in chickens.
Key words: Ascaridia galli, garlic, allicin, chicken, organic farming 2011 Poultry Science 90:364–368 doi:10.3382/ps.2010-01090
INTRODUCTION
ropean Union (CEC, 2007) and the philosophy behind this housing system have stimulated many farmers to search for and use alternatives to regular anthelmintics. One of these alternatives is garlic extract or powder, which is used as a water or feed supplement to control helminth infections. Garlic has been used for centuries to cure or prevent a large number of ailments. Many of these uses have been maintained through folklore and are now gaining the attention of scientists (Anthony et al., 2005; Tattelman, 2005). Various papers have described the antibacterial, antifungal, and antiprotozoal properties of garlic extracts (Dankert et al., 1979; Pai and Platt, 1995; Ankri and Mirelman, 1999; Harris et al., 2001; Davis, 2005; Ogita et al., 2005). The main active component of garlic is allicin, which is an organosulfur compound (diallyl thiosulfinate; Rabinkov et al., 1998; Ankri and Mirelman, 1999; Miron et al., 2000; Anthony et al., 2005). When a garlic bulb is damaged, separate segments containing the nonprotein amino acid alliin and the enzyme allinase are opened, resulting in the production of allicin in a rapid self-limiting reaction (Haciseferogullari et al., 2005). The antimicro-
Ascaridia galli is one of the most important helminth infections in poultry. A Danish study reported an estimated prevalence of 64% in free-range and organic poultry flocks and 42% in deep-litter systems (Permin et al., 1999). The most important clinical sign of A. galli infections is loss of BW, which increases parallel to worm load (Reid and Carmon, 1958). Increased feed intake (Gauly et al., 2007), blood loss, reduced BW gain, and increased mortality may also occur (Ikeme, 1971). Economic losses and welfare issues that result from severe A. galli infections are an important problem in laying flocks, especially in hens kept in free-range and organic farming systems (Ruff, 1999; Martín-Pacho et al., 2005). The obligatory outdoor pasture in these farming systems provides an ideal environment for the introduction, buildup, and maintenance of helminth infections. The organic agriculture regulation of the Eu©2011 Poultry Science Association Inc. Received August 30, 2010. Accepted November 9, 2010. 1 Corresponding author: [email protected]
364
RESEARCH NOTE
bial effects of allicin are explained by its ability to inhibit important thiol-dependent enzymatic systems and antioxidant activity, which have a multiple inhibitory effect on the microbial cell (Ankri and Mirelman, 1999; Anthony et al., 2005). Inhibition of the thiol-dependent enzymatic systems was found to be reversed by reactivation of the enzymes with thiol-containing compounds such as dithiothreitol, glutathione, and mercaptoethanol (Rabinkov et al., 1998). Compared with microbial cells, mammalian cells seem to be significantly less sensitive to the effects of allicin, which is most likely caused by the high concentrations of glutathione in the latter cells (Rabinkov et al., 1998). The effect of allicin on avian cells has not been described. However, because of the higher level of organization, avian cells are probably also less sensitive to allicin than microbes. Studies on the use of garlic preparations as anthelmintic strategies are scarce. A significant reduction of the Capillaria load in carp was shown after treatment of the water with garlic (Peña et al., 1988). It has been suggested in some manuscripts that garlic preparations have an effect on helminth infections in poultry. Administration of raw garlic (2.5 mg/bird) or an aqueous extract (2.5 mL/bird) daily for 5 d had some prophylactic action against A. galli infection in chickens (Das and Thakuria, 1974). In vitro tests showed that different concentrations of garlic oil (2, 4, and 6%) caused mortality of A. galli worms (Singh and Shalini, 2000). However, the therapeutic effect of allicin, a component of garlic, on helminth infections in poultry has not been described in the scientific literature to date. Therefore, a study was conducted to assess the effect of allicin from garlic on adult A. galli worms in chickens compared with anthelmintic treatment with flubendazole or no treatment.
MATERIALS AND METHODS
365
climatic conditions were set following the breeder’s recommendations. All bird losses (n = 13) during the experiment were unrelated to the A. galli infection and were attributed to cannibalism, as revealed by postmortem examination.
Preparation of Embryonated A. galli Eggs at Ridgeway Research Ltd. Chickens aged 7 to 14 d, kept at a Ridgeway Research Ltd. (St. Briavels, Gloucestershire, UK) facility, were each infected with 250 embryonated A. galli eggs by inoculation in the esophagus using a blunt needle at a volume of 0.5 mL/chicken. Birds were killed 6 wk later by cervical dislocation to harvest the worms from the intestinal tract. Worms were placed on a 150-µmpore-size sieve and washed several times with tap water. They were subsequently transferred to a food mixer (capacity 1 L; Moulinex, Paris, France) and homogenized for 10 to 20 s in a volume of 50 mL of water per 5 to 20 g of worms. The homogenate was successively passed through 75-, 53-, and 38-µm-pore-size sieves. The eggs were seen as a thin film on the 38-µm sieve, and they were washed off into a container with a wash bottle and were incubated at 27°C for 21 d in tap water at concentrations below 500 eggs/mL. The bottles containing the suspended eggs were shaken occasionally during incubation, and the water was changed at weekly intervals. The water change was performed as follows: the container was placed upright to allow the eggs to settle (2 to 3 h), and three-fourths of the total amount of water was siphoned off (using a water vacuum pump) and replaced with tap water. Eggs were stored at 10 to 25°C before use. A final suspension containing 430 embryonated eggs/mL was made with tap water.
Experimental Birds and Housing
Experimental Design
A total of 450 one-day-old Lohmann LSL-Classic cockerels were obtained from a commercial hatchery (Verbeek Broederij, Renswoude, the Netherlands) and were tagged for identification. They were fed a commercial premixed starter feed (De Heus Brokking Koudijs B.V., Ede, the Netherlands) containing 2,675 kcal of ME/kg and 17.5% CP, without coccidiostatic and antibiotic drugs, during the entire experiment. Feed and water were provided ad libitum. No vaccinations were given and chickens were not beak trimmed. Light was provided for 8 h/d. Up to 4 wk of age, birds received white light, and thereafter, red light was given at an intensity of approximately 6 lx to avert feather pecking and cannibalism. Until 5 wk of age, the chickens were kept as a single group, and thereafter, the chickens were randomly divided into 5 groups and housed in 2 identical separated rooms, each consisting of 3 identical floor pens with 1.5 kg of wood shavings/m2. During the entire experiment,
Group composition, inoculations, and treatments are outlined in Table 1. In short, at 6 wk of age, chickens from groups 2 to 5 were orally inoculated with A. galli eggs. Treatments were given to chickens from groups 3 to 5 from 13 wk of age onward. Group 1 (n = 50) served as the uninfected, untreated control group and group 2 (n = 93) served as the infected, untreated control group. Groups 3 to 5 (n = 98, 96, and 101, respectively) were infected and treated with the recommended dose of allicin (group 3), a 10-fold dose of allicin (group 4), or flubendazole (group 5). Groups 1 and 2 received tap water as a placebo treatment. At 13 wk of age, treatments were begun based on a positive fecal egg count of A. galli in groups 2 to 5. Twenty birds per infected group were necropsied weekly beginning just before the onset of treatments and continuing until the end of the experiment at 16 wk of age. In addition, 30 birds (10 birds at 13 wk of age and 20 birds at 16 wk of age) from group 1 were
366
Velkers et al.
Table 1. Design of the experiment to study the efficacy of allicin1 against Ascaridia galli infection in chickens: composition of groups, A. galli inoculation dose, and treatments
Group 1. 2. 3. 4. 5.
Uninfected, untreated control Infected, untreated control Allicin, single dose Allicin, 10-fold dose Flubendazole4
Chicks (no.)
A. galli eggs2 (no./bird)
50 93 98 96 101
None 300 300 300 300
Daily oral treatment3 (dose in 1 mL/bird) Tap water Tap water 225 µg of allicin in wk 1, 285 µg of allicin in wk 2 2,250 µg of allicin in wk 1, 2,850 µg of allicin in wk 2 12.5 mg of flubendazole in wk 1, tap water in wk 2
Duration of treatment (wk) 2 2 1, 1 1, 1 1, 1
1Allicin
International Ltd. (Rye, East Sussex, UK). with embryonated A. galli eggs in the esophagus was performed at 6 wk of age. 3Treatment began at 13 wk of age. 4Janssen Pharmaceuticals (Beerse, Belgium). 2Inoculation
selected at random and necropsied to confirm the absence of A. galli worms. All birds were killed by cervical dislocation for parasite isolation procedures.
Inocula for the Bird Experiment Birds of groups 2 to 5 were each inoculated in the esophagus with 300 embryonated A. galli eggs in a 0.7mL volume of tap water. The worm eggs were provided by Ridgeway Research Ltd. and were prepared as described in “Preparation of Embryonated A. galli Eggs at Ridgeway Research Ltd.”
Treatments A frozen allicin concentrate (5 mL/L) was obtained from Allicin International Ltd. (Rye, East Sussex, UK), from which a solution was made matching the allicin concentration recommended for use in the field (i.e., 1.5 mg/L of drinking water). Although the exact composition of the allicin concentrate used in this experiment was not available, analysis of allicin liquid concentrates by Halkes et al. (2008), using thin-layer chromatography, showed that allicin was the most prominent component. Further, small amounts of thiosulfinate and metabolites thereof and sulfur-containing amino acids were present. Considering expected daily water uptakes per chicken of 150 and 190 mL during the first and second weeks of treatment, respectively, based on the management guide from Lohmann Tierzucht GmbH (Cuxhaven, Germany), chickens from group 3 received a single recommended dose of 225 µg of allicin daily during the first week of treatment and 285 µg of allicin daily during the second week of treatment. Chickens from group 4 received a 10-fold dose (2,250 µg of allicin daily during the first week of treatment and 2,850 µg of allicin daily during the second week of treatment). Doses were given orally in 1 mL of tap water. A flubendazole suspension containing 12.5 mg of flubendazole/mL was made (Pharmacy of the Faculty of Veterinary Medicine, Utrecht University, the Netherlands). A commercially available, registered flubendazole 5% premix top dressing (Janssen Pharmaceuticals,
Beerse, Belgium) and sterile distilled water were used for preparation of the suspension. Mean BW of the birds at 13 wk of age was estimated at 1,250 g, based on prior BW measurements. To obtain a mean flubendazole dose of 10 mg/kg of BW, birds from group 5 received individual oral treatments with 1 mL of the flubendazole suspension daily for 7 d by using a 1-mL syringe. During the following week, each bird from this group received 1 mL of tap water without medication daily.
Parasite Isolation Procedures The digestive tract was excised and opened longitudinally from the gizzard to cloaca, including the ceca. Thereafter, the gut was placed in a large jar filled with tap water and was firmly shaken to remove all its contents. Subsequently, the digestive tract was removed from the jar and the contents of the jar were passed through a 100-µm-pore-size sieve. The retained worms were collected and counted on a Petri dish by visual observation.
Ethics All chickens were housed, handled, and treated following approval by the Animal Experimental Committee of Utrecht University, the Netherlands, in accordance with the Dutch regulation on experimental animals.
Statistical Analysis A GLM was applied using a negative binomial distribution to correct for the high variance to analyze the results of the worm counts (Lawless, 1987). The latter served as the dependent variable and the parameters “week of postmortem examination” and “group” were included in the model, as well as the interaction between these 2 variables. The infected, untreated control group was chosen as the reference group. The best model fit was assessed based on Akaike’s information criterion. All analyses were carried out with the statistical package R 2.2.0 (R Development Core Team, 2008).
367
RESEARCH NOTE
RESULTS Group 1 (uninfected, untreated control group) remained uninfected during the entire experiment. Table 2 gives the mean worm loads and SD per chicken for the other groups from 13 to 16 wk of age. At the beginning of treatment at 13 wk of age, the mean worm count in the flubendazole-treated group was slightly higher than that in the other groups but was not significantly different from that in the infected, untreated group (P ≥ 0.08). No significant differences were observed in worm counts in the allicin-treated groups (groups 3 and 4) compared with those in the infected, untreated group (group 2) in any week (P > 0.05). In contrast, worm counts were zero for the flubendazole-treated group (group 5) from 14 to 16 wk of age. A relatively low mean worm load was found in both allicin-treated groups (groups 3 and 4) at 15 wk of age (after 2 wk of treatment), but no significant difference from the infected, untreated group (group 2) was observed (P = 0.11 and P = 0.07 for groups 3 and 4, respectively).
DISCUSSION To determine whether treatment with allicin from garlic might be considered a potential alternative to deworming strategies using chemotherapeutical drugs, the intestinal adult worm load was used as a parameter to assess allicin efficacy in A. galli-infected chickens. Therefore, an experimental infection was induced in 6-wk-old cockerels, resulting in a mean adult worm load of approximately 16 worms/chicken at 13 wk of age. Although all inoculated chickens received the same number of embryonated A. galli eggs (300/chicken), great differences in worm load were found between birds. This finding is consistent with other experimentally induced A. galli infections (Permin et al., 1997; Gauly et al., 2001, 2005; Idi et al., 2004). In our study, flubendazole treatment completely eliminated the induced worm infection, whereas allicin did not.
In this experiment, all birds were treated individually, instead of via drinking water, to standardize the intake of allicin and flubendazole. Although drinking water medication is the prescribed method for using allicin, it is likely that individual bolus treatment would be equally as effective as, or even more effective than, drinking water medication, all the more because a 10fold dose was administered by bolus. However, even the 10-fold dose of allicin per bolus had no significant effect on the number of adult A. galli worms. Effects of allicin treatment on larval development of A. galli in the intestinal mucosa or on BW development, feed intake, feed conversion, and egg production were not determined in this study. Although some authors (Reid and Carmon, 1958; Gauly et al., 2002, 2007) and farmers have reported effects on these parameters because of the presence of adult or larval A. galli stages, these parameters were irrelevant regarding the primary aim of the current study, namely, to assess the efficacy of allicin on the A. galli worm load in chickens compared with standard deworming strategies with flubendazole. However, these parameters might be considered in studies aimed toward determining whether allicin treatments might have any beneficial effects on production and performance in A. galli-infected layer flocks. The size of the experimental groups was large enough to detect significant differences in worm load between the flubendazole-treated birds and infected, untreated cockerels. However, because of large differences in worm counts, the statistical power was insufficient to detect small effects of allicin treatment on the adult worm counts. Nevertheless, because the aim of the study was to determine whether allicin treatment could potentially substitute for flubendazole in the treatment of A. galli infections in organic chicken farming, the group sizes used in the present study sufficed. It can therefore be concluded that allicin liquid at the doses examined cannot be used as a replacement for flubendazole treatment. Several studies have suggested that allicin is the active compound in garlic (Rabinkov et al., 1998; Ankri
Table 2. Mean number of adult Ascaridia galli worms (±SD) in the intestinal tract of experimentally infected chickens for each experimental group Age (wk) 136 14 15 16 1Infection
Birds (no.) 20 20 20 20
Group 2: infected,1 untreated control 13.8 12.8 12.9 11.9
± ± ± ±
12.0 9.9 9.2 10.8
Group 3: infected,1 allicin2 treatment, single dose3 12.9 11.4 8.1 12.3
± ± ± ±
10.1 12.6 6.7 11.8
Group 4: infected,1 allicin2 treatment, 10-fold dose4 15.0 13.5 8.9 10.8
± ± ± ±
11.2 11.0 6.6 9.2
Group 5: infected,1 flubendazole treatment5 22.5 ± 14.8 0 0 0
with 300 embryonated A. galli eggs was performed at 6 wk of age. International Ltd. (Rye, East Sussex, UK). 3Dose in wk 1 of treatment was 225 µg of allicin/bird and in wk 2 of treatment was 285 µg of allicin/bird. 4Dose in wk 1 of treatment was 2,250 µg of allicin/bird and in wk 2 was 2,850 µg of allicin/bird. 5Dose in wk 1 of treatment was 12.5 mg of flubendazole/bird (Janssen Pharmaceuticals, Beerse, Belgium) and in wk 2 of treatment was 1 mL of tap water/bird. 6Beginning of treatment. 2Allicin
368
Velkers et al.
and Mirelman, 1999; Miron et al., 2000; Harris et al., 2001). Although the manufacturer of the allicin liquid claims that allicin has been stabilized in this product by using a patented process, Amagase (2006) argued that allicin is highly unstable in commercial preparations and that other constituents of garlic could be the “real bioactive constituents of garlic.” Therefore, it cannot be concluded that garlic products other than allicin are also ineffective against A. galli infestations. Further research into the efficacy of other active components of garlic is necessary to elucidate this.
ACKNOWLEDGMENTS The authors thank Maarten Eysker from the Department of Infectious Diseases and Immunology, Clinical Infectiology Division of the Faculty of Veterinary Medicine (Utrecht, the Netherlands) and Rainier van Gelderen of Janssen Animal Health (Beerse, Belgium) for their input in the experimental design. In addition, we thank the biotechnicians of the experimental animal facility at the Department of Farm Animal Health of the Faculty of Veterinary Medicine (Utrecht, the Netherlands) for their contribution to the experiments.
REFERENCES Amagase, H. 2006. Clarifying the real bioactive constituents of garlic. J. Nutr. 136:716S–725S. Ankri, S., and D. Mirelman. 1999. Antimicrobial properties of allicin from garlic. Microbes Infect. 1:125–129. Anthony, J. P., L. Fyfe, and H. Smith. 2005. Plant active components—A resource for antiparasitic agents? Trends Parasitol. 21:462–468. CEC (Commission of the European Communities). 2007. On organic production and labelling of organic products and repealing regulation. Off. J. Eur. Commun. L 189:1–23. Dankert, J., T. F. Tromp, H. de Vries, and H. J. Klasen. 1979. Antimicrobial activity of crude juices of Allium ascalonicum, Allium cepa and Allium sativum. Zentralbl. Bakteriol. (Orig. A) 245:229–239. Das, P. N., and B. N. Thakuria. 1974. Anthelmintic effect of garlic (Allium sativum) against Ascaridia galli. Vetcol.: J. Coll. Vet. Sci. (Assam Agric. Univ.) 14:47–52. Davis, S. R. 2005. An overview of the antifungal properties of allicin and its breakdown products—The possibility of a safe and effective antifungal prophylactic. Mycoses 48:95–100. Gauly, M., C. Bauer, C. Mertens, and G. Erhardt. 2001. Effect and repeatability of Ascaridia galli egg output in cockerels following a single low dose infection. Vet. Parasitol. 96:301–307. Gauly, M., C. Bauer, R. Preisinger, and G. Erhardt. 2002. Genetic differences of Ascaridia galli egg output in laying hens following a single dose infection. Vet. Parasitol. 103:99–107. Gauly, M., C. Duss, and G. Erhardt. 2007. Influence of Ascaridia galli infections and anthelmintic treatments on the behaviour and social ranks of laying hens (Gallus gallus domesticus). Vet. Parasitol. 146:271–280. Gauly, M., T. Homann, and G. Erhardt. 2005. Age-related differences of Ascaridia galli egg output and worm burden in chickens following a single dose infection. Vet. Parasitol. 128:141–148.
Haciseferogullari, H., M. Ozcan, F. Demir, and S. Calisir. 2005. Some nutritional and technological properties of garlic (Allium sativum L.). J. Food Eng. 68:463–469. Halkes, S. B. A., L. Quarles van Ufford, E. Worm, C. J. Beukelman, A. J. J. van den Berg. 2008. Eindrapport Fyto V, Annex 2A: Toetsing van geselecteerde preparaten; kwaliteitscontrole en in vitro biologische activiteit. Rikilt Institute of Food Safety. Accessed March 2010. http://www.rikilt.wur.nl/NL/publicaties/ Rapporten/Fyto+V/. Harris, J. C., S. L. Cottrell, S. Plummer, and D. Lloyd. 2001. Antimicrobial properties of Allium sativum (garlic). Appl. Microbiol. Biotechnol. 57:282–286. Idi, A., A. Permin, and K. D. Murrell. 2004. Host age only partially affects resistance to primary and secondary infections with Ascaridia galli (Schrank, 1788) in chickens. Vet. Parasitol. 122:221–231. Ikeme, M. M. 1971. Observations on the pathogenicity and pathology of Ascaridia galli. Parasitology 63:169–179. Lawless, J. F. 1987. Negative binomial and mixed Poisson regression. Can. J. Stat. 15:209–225. Martín-Pacho, J. R., M. N. Montoya, T. Aranguena, C. Toro, R. Morchon, C. Marcos-Atxutegi, and F. Simon. 2005. A coprological and serological survey for the prevalence of Ascaridia spp. in laying hens. J. Vet. Med. B Infect. Dis. Vet. Public Health 52:238–242. Miron, T., A. Rabinkov, D. Mirelman, M. Wilchek, and L. Weiner. 2000. The mode of action of allicin: Its ready permeability through phospholipid membranes may contribute to its biological activity. Biochim. Biophys. Acta 1463:20–30. Ogita, A., K. Hirooka, Y. Yamamoto, N. Tsutsui, K. Fujita, M. Taniguchi, and T. Tanaka. 2005. Synergistic fungicidal activity of Cu2+ and allicin, an allyl sulfur compound from garlic, and its relation to the role of alkyl hydroperoxide reductase 1 as a cell surface defense in Saccharomyces cerevisiae. Toxicology 215:205–213. Pai, S. T., and M. W. Platt. 1995. Antifungal effects of Allium sativum (garlic) extract against the Aspergillus species involved in otomycosis. Lett. Appl. Microbiol. 20:14–18. Peña, N., A. Auro, and H. Sumano. 1988. A comparative trial of garlic, its extract and ammonium-potassium tartrate as anthelmintics in carp. J. Ethnopharmacol. 24:199–203. Permin, A., M. Bisgaard, F. Frandsen, M. Pearman, J. Kold, and P. Nansen. 1999. Prevalence of gastrointestinal helminths in different poultry production systems. Br. Poult. Sci. 40:439–443. Permin, A., M. Bojesen, P. Nansen, M. Bisgaard, F. Frandsen, and M. Pearman. 1997. Ascaridia galli populations in chickens following single infections with different dose levels. Parasitol. Res. 83:614–617. Rabinkov, A., T. Miron, L. Konstantinovski, M. Wilchek, D. Mirelman, and L. Weiner. 1998. The mode of action of allicin: Trapping of radicals and interaction with thiol containing proteins. Biochim. Biophys. Acta 1379:233–244. R Development Core Team. 2008. R: A language and environment for statistical computing. The R Foundation for Statistical Computing, Vienna, Austria. Accessed March 2010. http://www.rproject.org/. Reid, W. M., and J. L. Carmon. 1958. Effects of numbers of Ascaridia galli in depressing weight gains in chicks. J. Parasitol. 44:183–186. Ruff, M. D. 1999. Important parasites in poultry production systems. Vet. Parasitol. 84:337–347. Singh, K., and N. Shalini. 2000. Studies on the anthelmintic activity of Allium sativum (garlic) oil on common poultry worms Ascaridia galli and Heterakis gallinae. J. Parasitol. Appl. Anim. Biol. 9:47–52. Tattelman, E. 2005. Health effects of garlic. Am. Fam. Physician 72:103–106.
Group 3 received the recommended dose of allicin for 2 wk, whereas group 4 received a 10-fold dose of allicin. Group 5 was given 10 mg of flubendazole/kg of BW for 1 wk. Necropsy of 20 birds of all groups was performed weekly between 13 and 16 wk of age to determine adult worm loads. Group 1 remained free of A. galli. The experimental infection in the other groups resulted in a mean adult worm load of approximately 16 worms/ bird. No significant differences were observed in worm counts of the allicin-treated groups (groups 3 and 4) compared with the infected, untreated group (group 2) at any week (P > 0.05). In contrast, no worms were found in chickens after flubendazole treatment (group 5). It was concluded that allicin does not represent an alternative to flubendazole for the treatment of A. galli infections in chickens.
Key words: Ascaridia galli, garlic, allicin, chicken, organic farming 2011 Poultry Science 90:364–368 doi:10.3382/ps.2010-01090
INTRODUCTION
ropean Union (CEC, 2007) and the philosophy behind this housing system have stimulated many farmers to search for and use alternatives to regular anthelmintics. One of these alternatives is garlic extract or powder, which is used as a water or feed supplement to control helminth infections. Garlic has been used for centuries to cure or prevent a large number of ailments. Many of these uses have been maintained through folklore and are now gaining the attention of scientists (Anthony et al., 2005; Tattelman, 2005). Various papers have described the antibacterial, antifungal, and antiprotozoal properties of garlic extracts (Dankert et al., 1979; Pai and Platt, 1995; Ankri and Mirelman, 1999; Harris et al., 2001; Davis, 2005; Ogita et al., 2005). The main active component of garlic is allicin, which is an organosulfur compound (diallyl thiosulfinate; Rabinkov et al., 1998; Ankri and Mirelman, 1999; Miron et al., 2000; Anthony et al., 2005). When a garlic bulb is damaged, separate segments containing the nonprotein amino acid alliin and the enzyme allinase are opened, resulting in the production of allicin in a rapid self-limiting reaction (Haciseferogullari et al., 2005). The antimicro-
Ascaridia galli is one of the most important helminth infections in poultry. A Danish study reported an estimated prevalence of 64% in free-range and organic poultry flocks and 42% in deep-litter systems (Permin et al., 1999). The most important clinical sign of A. galli infections is loss of BW, which increases parallel to worm load (Reid and Carmon, 1958). Increased feed intake (Gauly et al., 2007), blood loss, reduced BW gain, and increased mortality may also occur (Ikeme, 1971). Economic losses and welfare issues that result from severe A. galli infections are an important problem in laying flocks, especially in hens kept in free-range and organic farming systems (Ruff, 1999; Martín-Pacho et al., 2005). The obligatory outdoor pasture in these farming systems provides an ideal environment for the introduction, buildup, and maintenance of helminth infections. The organic agriculture regulation of the Eu©2011 Poultry Science Association Inc. Received August 30, 2010. Accepted November 9, 2010. 1 Corresponding author: [email protected]
364
RESEARCH NOTE
bial effects of allicin are explained by its ability to inhibit important thiol-dependent enzymatic systems and antioxidant activity, which have a multiple inhibitory effect on the microbial cell (Ankri and Mirelman, 1999; Anthony et al., 2005). Inhibition of the thiol-dependent enzymatic systems was found to be reversed by reactivation of the enzymes with thiol-containing compounds such as dithiothreitol, glutathione, and mercaptoethanol (Rabinkov et al., 1998). Compared with microbial cells, mammalian cells seem to be significantly less sensitive to the effects of allicin, which is most likely caused by the high concentrations of glutathione in the latter cells (Rabinkov et al., 1998). The effect of allicin on avian cells has not been described. However, because of the higher level of organization, avian cells are probably also less sensitive to allicin than microbes. Studies on the use of garlic preparations as anthelmintic strategies are scarce. A significant reduction of the Capillaria load in carp was shown after treatment of the water with garlic (Peña et al., 1988). It has been suggested in some manuscripts that garlic preparations have an effect on helminth infections in poultry. Administration of raw garlic (2.5 mg/bird) or an aqueous extract (2.5 mL/bird) daily for 5 d had some prophylactic action against A. galli infection in chickens (Das and Thakuria, 1974). In vitro tests showed that different concentrations of garlic oil (2, 4, and 6%) caused mortality of A. galli worms (Singh and Shalini, 2000). However, the therapeutic effect of allicin, a component of garlic, on helminth infections in poultry has not been described in the scientific literature to date. Therefore, a study was conducted to assess the effect of allicin from garlic on adult A. galli worms in chickens compared with anthelmintic treatment with flubendazole or no treatment.
MATERIALS AND METHODS
365
climatic conditions were set following the breeder’s recommendations. All bird losses (n = 13) during the experiment were unrelated to the A. galli infection and were attributed to cannibalism, as revealed by postmortem examination.
Preparation of Embryonated A. galli Eggs at Ridgeway Research Ltd. Chickens aged 7 to 14 d, kept at a Ridgeway Research Ltd. (St. Briavels, Gloucestershire, UK) facility, were each infected with 250 embryonated A. galli eggs by inoculation in the esophagus using a blunt needle at a volume of 0.5 mL/chicken. Birds were killed 6 wk later by cervical dislocation to harvest the worms from the intestinal tract. Worms were placed on a 150-µmpore-size sieve and washed several times with tap water. They were subsequently transferred to a food mixer (capacity 1 L; Moulinex, Paris, France) and homogenized for 10 to 20 s in a volume of 50 mL of water per 5 to 20 g of worms. The homogenate was successively passed through 75-, 53-, and 38-µm-pore-size sieves. The eggs were seen as a thin film on the 38-µm sieve, and they were washed off into a container with a wash bottle and were incubated at 27°C for 21 d in tap water at concentrations below 500 eggs/mL. The bottles containing the suspended eggs were shaken occasionally during incubation, and the water was changed at weekly intervals. The water change was performed as follows: the container was placed upright to allow the eggs to settle (2 to 3 h), and three-fourths of the total amount of water was siphoned off (using a water vacuum pump) and replaced with tap water. Eggs were stored at 10 to 25°C before use. A final suspension containing 430 embryonated eggs/mL was made with tap water.
Experimental Birds and Housing
Experimental Design
A total of 450 one-day-old Lohmann LSL-Classic cockerels were obtained from a commercial hatchery (Verbeek Broederij, Renswoude, the Netherlands) and were tagged for identification. They were fed a commercial premixed starter feed (De Heus Brokking Koudijs B.V., Ede, the Netherlands) containing 2,675 kcal of ME/kg and 17.5% CP, without coccidiostatic and antibiotic drugs, during the entire experiment. Feed and water were provided ad libitum. No vaccinations were given and chickens were not beak trimmed. Light was provided for 8 h/d. Up to 4 wk of age, birds received white light, and thereafter, red light was given at an intensity of approximately 6 lx to avert feather pecking and cannibalism. Until 5 wk of age, the chickens were kept as a single group, and thereafter, the chickens were randomly divided into 5 groups and housed in 2 identical separated rooms, each consisting of 3 identical floor pens with 1.5 kg of wood shavings/m2. During the entire experiment,
Group composition, inoculations, and treatments are outlined in Table 1. In short, at 6 wk of age, chickens from groups 2 to 5 were orally inoculated with A. galli eggs. Treatments were given to chickens from groups 3 to 5 from 13 wk of age onward. Group 1 (n = 50) served as the uninfected, untreated control group and group 2 (n = 93) served as the infected, untreated control group. Groups 3 to 5 (n = 98, 96, and 101, respectively) were infected and treated with the recommended dose of allicin (group 3), a 10-fold dose of allicin (group 4), or flubendazole (group 5). Groups 1 and 2 received tap water as a placebo treatment. At 13 wk of age, treatments were begun based on a positive fecal egg count of A. galli in groups 2 to 5. Twenty birds per infected group were necropsied weekly beginning just before the onset of treatments and continuing until the end of the experiment at 16 wk of age. In addition, 30 birds (10 birds at 13 wk of age and 20 birds at 16 wk of age) from group 1 were
366
Velkers et al.
Table 1. Design of the experiment to study the efficacy of allicin1 against Ascaridia galli infection in chickens: composition of groups, A. galli inoculation dose, and treatments
Group 1. 2. 3. 4. 5.
Uninfected, untreated control Infected, untreated control Allicin, single dose Allicin, 10-fold dose Flubendazole4
Chicks (no.)
A. galli eggs2 (no./bird)
50 93 98 96 101
None 300 300 300 300
Daily oral treatment3 (dose in 1 mL/bird) Tap water Tap water 225 µg of allicin in wk 1, 285 µg of allicin in wk 2 2,250 µg of allicin in wk 1, 2,850 µg of allicin in wk 2 12.5 mg of flubendazole in wk 1, tap water in wk 2
Duration of treatment (wk) 2 2 1, 1 1, 1 1, 1
1Allicin
International Ltd. (Rye, East Sussex, UK). with embryonated A. galli eggs in the esophagus was performed at 6 wk of age. 3Treatment began at 13 wk of age. 4Janssen Pharmaceuticals (Beerse, Belgium). 2Inoculation
selected at random and necropsied to confirm the absence of A. galli worms. All birds were killed by cervical dislocation for parasite isolation procedures.
Inocula for the Bird Experiment Birds of groups 2 to 5 were each inoculated in the esophagus with 300 embryonated A. galli eggs in a 0.7mL volume of tap water. The worm eggs were provided by Ridgeway Research Ltd. and were prepared as described in “Preparation of Embryonated A. galli Eggs at Ridgeway Research Ltd.”
Treatments A frozen allicin concentrate (5 mL/L) was obtained from Allicin International Ltd. (Rye, East Sussex, UK), from which a solution was made matching the allicin concentration recommended for use in the field (i.e., 1.5 mg/L of drinking water). Although the exact composition of the allicin concentrate used in this experiment was not available, analysis of allicin liquid concentrates by Halkes et al. (2008), using thin-layer chromatography, showed that allicin was the most prominent component. Further, small amounts of thiosulfinate and metabolites thereof and sulfur-containing amino acids were present. Considering expected daily water uptakes per chicken of 150 and 190 mL during the first and second weeks of treatment, respectively, based on the management guide from Lohmann Tierzucht GmbH (Cuxhaven, Germany), chickens from group 3 received a single recommended dose of 225 µg of allicin daily during the first week of treatment and 285 µg of allicin daily during the second week of treatment. Chickens from group 4 received a 10-fold dose (2,250 µg of allicin daily during the first week of treatment and 2,850 µg of allicin daily during the second week of treatment). Doses were given orally in 1 mL of tap water. A flubendazole suspension containing 12.5 mg of flubendazole/mL was made (Pharmacy of the Faculty of Veterinary Medicine, Utrecht University, the Netherlands). A commercially available, registered flubendazole 5% premix top dressing (Janssen Pharmaceuticals,
Beerse, Belgium) and sterile distilled water were used for preparation of the suspension. Mean BW of the birds at 13 wk of age was estimated at 1,250 g, based on prior BW measurements. To obtain a mean flubendazole dose of 10 mg/kg of BW, birds from group 5 received individual oral treatments with 1 mL of the flubendazole suspension daily for 7 d by using a 1-mL syringe. During the following week, each bird from this group received 1 mL of tap water without medication daily.
Parasite Isolation Procedures The digestive tract was excised and opened longitudinally from the gizzard to cloaca, including the ceca. Thereafter, the gut was placed in a large jar filled with tap water and was firmly shaken to remove all its contents. Subsequently, the digestive tract was removed from the jar and the contents of the jar were passed through a 100-µm-pore-size sieve. The retained worms were collected and counted on a Petri dish by visual observation.
Ethics All chickens were housed, handled, and treated following approval by the Animal Experimental Committee of Utrecht University, the Netherlands, in accordance with the Dutch regulation on experimental animals.
Statistical Analysis A GLM was applied using a negative binomial distribution to correct for the high variance to analyze the results of the worm counts (Lawless, 1987). The latter served as the dependent variable and the parameters “week of postmortem examination” and “group” were included in the model, as well as the interaction between these 2 variables. The infected, untreated control group was chosen as the reference group. The best model fit was assessed based on Akaike’s information criterion. All analyses were carried out with the statistical package R 2.2.0 (R Development Core Team, 2008).
367
RESEARCH NOTE
RESULTS Group 1 (uninfected, untreated control group) remained uninfected during the entire experiment. Table 2 gives the mean worm loads and SD per chicken for the other groups from 13 to 16 wk of age. At the beginning of treatment at 13 wk of age, the mean worm count in the flubendazole-treated group was slightly higher than that in the other groups but was not significantly different from that in the infected, untreated group (P ≥ 0.08). No significant differences were observed in worm counts in the allicin-treated groups (groups 3 and 4) compared with those in the infected, untreated group (group 2) in any week (P > 0.05). In contrast, worm counts were zero for the flubendazole-treated group (group 5) from 14 to 16 wk of age. A relatively low mean worm load was found in both allicin-treated groups (groups 3 and 4) at 15 wk of age (after 2 wk of treatment), but no significant difference from the infected, untreated group (group 2) was observed (P = 0.11 and P = 0.07 for groups 3 and 4, respectively).
DISCUSSION To determine whether treatment with allicin from garlic might be considered a potential alternative to deworming strategies using chemotherapeutical drugs, the intestinal adult worm load was used as a parameter to assess allicin efficacy in A. galli-infected chickens. Therefore, an experimental infection was induced in 6-wk-old cockerels, resulting in a mean adult worm load of approximately 16 worms/chicken at 13 wk of age. Although all inoculated chickens received the same number of embryonated A. galli eggs (300/chicken), great differences in worm load were found between birds. This finding is consistent with other experimentally induced A. galli infections (Permin et al., 1997; Gauly et al., 2001, 2005; Idi et al., 2004). In our study, flubendazole treatment completely eliminated the induced worm infection, whereas allicin did not.
In this experiment, all birds were treated individually, instead of via drinking water, to standardize the intake of allicin and flubendazole. Although drinking water medication is the prescribed method for using allicin, it is likely that individual bolus treatment would be equally as effective as, or even more effective than, drinking water medication, all the more because a 10fold dose was administered by bolus. However, even the 10-fold dose of allicin per bolus had no significant effect on the number of adult A. galli worms. Effects of allicin treatment on larval development of A. galli in the intestinal mucosa or on BW development, feed intake, feed conversion, and egg production were not determined in this study. Although some authors (Reid and Carmon, 1958; Gauly et al., 2002, 2007) and farmers have reported effects on these parameters because of the presence of adult or larval A. galli stages, these parameters were irrelevant regarding the primary aim of the current study, namely, to assess the efficacy of allicin on the A. galli worm load in chickens compared with standard deworming strategies with flubendazole. However, these parameters might be considered in studies aimed toward determining whether allicin treatments might have any beneficial effects on production and performance in A. galli-infected layer flocks. The size of the experimental groups was large enough to detect significant differences in worm load between the flubendazole-treated birds and infected, untreated cockerels. However, because of large differences in worm counts, the statistical power was insufficient to detect small effects of allicin treatment on the adult worm counts. Nevertheless, because the aim of the study was to determine whether allicin treatment could potentially substitute for flubendazole in the treatment of A. galli infections in organic chicken farming, the group sizes used in the present study sufficed. It can therefore be concluded that allicin liquid at the doses examined cannot be used as a replacement for flubendazole treatment. Several studies have suggested that allicin is the active compound in garlic (Rabinkov et al., 1998; Ankri
Table 2. Mean number of adult Ascaridia galli worms (±SD) in the intestinal tract of experimentally infected chickens for each experimental group Age (wk) 136 14 15 16 1Infection
Birds (no.) 20 20 20 20
Group 2: infected,1 untreated control 13.8 12.8 12.9 11.9
± ± ± ±
12.0 9.9 9.2 10.8
Group 3: infected,1 allicin2 treatment, single dose3 12.9 11.4 8.1 12.3
± ± ± ±
10.1 12.6 6.7 11.8
Group 4: infected,1 allicin2 treatment, 10-fold dose4 15.0 13.5 8.9 10.8
± ± ± ±
11.2 11.0 6.6 9.2
Group 5: infected,1 flubendazole treatment5 22.5 ± 14.8 0 0 0
with 300 embryonated A. galli eggs was performed at 6 wk of age. International Ltd. (Rye, East Sussex, UK). 3Dose in wk 1 of treatment was 225 µg of allicin/bird and in wk 2 of treatment was 285 µg of allicin/bird. 4Dose in wk 1 of treatment was 2,250 µg of allicin/bird and in wk 2 was 2,850 µg of allicin/bird. 5Dose in wk 1 of treatment was 12.5 mg of flubendazole/bird (Janssen Pharmaceuticals, Beerse, Belgium) and in wk 2 of treatment was 1 mL of tap water/bird. 6Beginning of treatment. 2Allicin
368
Velkers et al.
and Mirelman, 1999; Miron et al., 2000; Harris et al., 2001). Although the manufacturer of the allicin liquid claims that allicin has been stabilized in this product by using a patented process, Amagase (2006) argued that allicin is highly unstable in commercial preparations and that other constituents of garlic could be the “real bioactive constituents of garlic.” Therefore, it cannot be concluded that garlic products other than allicin are also ineffective against A. galli infestations. Further research into the efficacy of other active components of garlic is necessary to elucidate this.
ACKNOWLEDGMENTS The authors thank Maarten Eysker from the Department of Infectious Diseases and Immunology, Clinical Infectiology Division of the Faculty of Veterinary Medicine (Utrecht, the Netherlands) and Rainier van Gelderen of Janssen Animal Health (Beerse, Belgium) for their input in the experimental design. In addition, we thank the biotechnicians of the experimental animal facility at the Department of Farm Animal Health of the Faculty of Veterinary Medicine (Utrecht, the Netherlands) for their contribution to the experiments.
REFERENCES Amagase, H. 2006. Clarifying the real bioactive constituents of garlic. J. Nutr. 136:716S–725S. Ankri, S., and D. Mirelman. 1999. Antimicrobial properties of allicin from garlic. Microbes Infect. 1:125–129. Anthony, J. P., L. Fyfe, and H. Smith. 2005. Plant active components—A resource for antiparasitic agents? Trends Parasitol. 21:462–468. CEC (Commission of the European Communities). 2007. On organic production and labelling of organic products and repealing regulation. Off. J. Eur. Commun. L 189:1–23. Dankert, J., T. F. Tromp, H. de Vries, and H. J. Klasen. 1979. Antimicrobial activity of crude juices of Allium ascalonicum, Allium cepa and Allium sativum. Zentralbl. Bakteriol. (Orig. A) 245:229–239. Das, P. N., and B. N. Thakuria. 1974. Anthelmintic effect of garlic (Allium sativum) against Ascaridia galli. Vetcol.: J. Coll. Vet. Sci. (Assam Agric. Univ.) 14:47–52. Davis, S. R. 2005. An overview of the antifungal properties of allicin and its breakdown products—The possibility of a safe and effective antifungal prophylactic. Mycoses 48:95–100. Gauly, M., C. Bauer, C. Mertens, and G. Erhardt. 2001. Effect and repeatability of Ascaridia galli egg output in cockerels following a single low dose infection. Vet. Parasitol. 96:301–307. Gauly, M., C. Bauer, R. Preisinger, and G. Erhardt. 2002. Genetic differences of Ascaridia galli egg output in laying hens following a single dose infection. Vet. Parasitol. 103:99–107. Gauly, M., C. Duss, and G. Erhardt. 2007. Influence of Ascaridia galli infections and anthelmintic treatments on the behaviour and social ranks of laying hens (Gallus gallus domesticus). Vet. Parasitol. 146:271–280. Gauly, M., T. Homann, and G. Erhardt. 2005. Age-related differences of Ascaridia galli egg output and worm burden in chickens following a single dose infection. Vet. Parasitol. 128:141–148.
Haciseferogullari, H., M. Ozcan, F. Demir, and S. Calisir. 2005. Some nutritional and technological properties of garlic (Allium sativum L.). J. Food Eng. 68:463–469. Halkes, S. B. A., L. Quarles van Ufford, E. Worm, C. J. Beukelman, A. J. J. van den Berg. 2008. Eindrapport Fyto V, Annex 2A: Toetsing van geselecteerde preparaten; kwaliteitscontrole en in vitro biologische activiteit. Rikilt Institute of Food Safety. Accessed March 2010. http://www.rikilt.wur.nl/NL/publicaties/ Rapporten/Fyto+V/. Harris, J. C., S. L. Cottrell, S. Plummer, and D. Lloyd. 2001. Antimicrobial properties of Allium sativum (garlic). Appl. Microbiol. Biotechnol. 57:282–286. Idi, A., A. Permin, and K. D. Murrell. 2004. Host age only partially affects resistance to primary and secondary infections with Ascaridia galli (Schrank, 1788) in chickens. Vet. Parasitol. 122:221–231. Ikeme, M. M. 1971. Observations on the pathogenicity and pathology of Ascaridia galli. Parasitology 63:169–179. Lawless, J. F. 1987. Negative binomial and mixed Poisson regression. Can. J. Stat. 15:209–225. Martín-Pacho, J. R., M. N. Montoya, T. Aranguena, C. Toro, R. Morchon, C. Marcos-Atxutegi, and F. Simon. 2005. A coprological and serological survey for the prevalence of Ascaridia spp. in laying hens. J. Vet. Med. B Infect. Dis. Vet. Public Health 52:238–242. Miron, T., A. Rabinkov, D. Mirelman, M. Wilchek, and L. Weiner. 2000. The mode of action of allicin: Its ready permeability through phospholipid membranes may contribute to its biological activity. Biochim. Biophys. Acta 1463:20–30. Ogita, A., K. Hirooka, Y. Yamamoto, N. Tsutsui, K. Fujita, M. Taniguchi, and T. Tanaka. 2005. Synergistic fungicidal activity of Cu2+ and allicin, an allyl sulfur compound from garlic, and its relation to the role of alkyl hydroperoxide reductase 1 as a cell surface defense in Saccharomyces cerevisiae. Toxicology 215:205–213. Pai, S. T., and M. W. Platt. 1995. Antifungal effects of Allium sativum (garlic) extract against the Aspergillus species involved in otomycosis. Lett. Appl. Microbiol. 20:14–18. Peña, N., A. Auro, and H. Sumano. 1988. A comparative trial of garlic, its extract and ammonium-potassium tartrate as anthelmintics in carp. J. Ethnopharmacol. 24:199–203. Permin, A., M. Bisgaard, F. Frandsen, M. Pearman, J. Kold, and P. Nansen. 1999. Prevalence of gastrointestinal helminths in different poultry production systems. Br. Poult. Sci. 40:439–443. Permin, A., M. Bojesen, P. Nansen, M. Bisgaard, F. Frandsen, and M. Pearman. 1997. Ascaridia galli populations in chickens following single infections with different dose levels. Parasitol. Res. 83:614–617. Rabinkov, A., T. Miron, L. Konstantinovski, M. Wilchek, D. Mirelman, and L. Weiner. 1998. The mode of action of allicin: Trapping of radicals and interaction with thiol containing proteins. Biochim. Biophys. Acta 1379:233–244. R Development Core Team. 2008. R: A language and environment for statistical computing. The R Foundation for Statistical Computing, Vienna, Austria. Accessed March 2010. http://www.rproject.org/. Reid, W. M., and J. L. Carmon. 1958. Effects of numbers of Ascaridia galli in depressing weight gains in chicks. J. Parasitol. 44:183–186. Ruff, M. D. 1999. Important parasites in poultry production systems. Vet. Parasitol. 84:337–347. Singh, K., and N. Shalini. 2000. Studies on the anthelmintic activity of Allium sativum (garlic) oil on common poultry worms Ascaridia galli and Heterakis gallinae. J. Parasitol. Appl. Anim. Biol. 9:47–52. Tattelman, E. 2005. Health effects of garlic. Am. Fam. Physician 72:103–106.