Parasitol Res (2012) 111:22292233 DOI 10.

1007/s00436-012-3071-y

ORIGINAL PAPER

Anticoccidial activity of traditional Chinese herbal Dichroa febrifuga Lour. extract against Eimeria tenella infection in chickens
De-Fu Zhang & Bing-Bing Sun & Ying-Ying Yue & Qian-Jin Zhou & Ai-Fang Du

Received: 27 April 2012 / Accepted: 30 July 2012 / Published online: 17 August 2012 # Springer-Verlag 2012

Abstract The study was conducted on broiler birds to evaluate the anticoccidial efficacy of an extract of Chinese traditional herb Dichroa febrifuga Lour. One hundred broiler birds were assigned to five equal groups. All birds in groups 14 were orally infected with 1.5104 Eimeira tenella sporulated oocysts and birds in groups 1, 2 and 3 were medicated with 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respectively. The bloody diarrhea, oocyst counts, intestinal lesion scores, and the body weight were recorded to evaluate the anticoccidial efficacy. The results showed that D. febrifuga extract was effective against Eimeria infection; especially 20 mg D. febrifuga extract/kg feed can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion in comparison to infected-unmedicated control group.

Introduction Coccidiosis may cause severe intestinal diseases. Coccidiosis is distributed worldwide in poultry and wild birds and is of major economic impact in poultry production due to mortality and morbidity. Epidemiologic studies have shown that the mortality of fowl can range from 5 to 70 % (Du and Hu 2004; Michels et al. 2011). Economic losses caused by avian coccidiosis, including decreased productivity, usage of coccidiostat, and vaccines, are estimated to amount to $3 billion annually (Dalloul and Lillehoj 2006; Lai et al. 2011; Williams 1999; Zhang et al. 2012b; Michels et al. 2011). In China alone, the estimated expenditure is about $3060 million due to the
D.-F. Zhang : B.-B. Sun : Y.-Y. Yue : Q.-J. Zhou : A.-F. Du (*) Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Peoples Republic of China e-mail: afdu@zju.edu.cn

use of anticoccidial drugs (Hao et al. 2007). The domestic poultry industry of People's Republic of China primarily relies on medical prophylaxis. But the emergence of problems related to drug resistance and drug residues of antibiotics in the chicken meat has stimulated us to seek safer and more efficacious alternative control strategies (Lai et al. 2011). Chinese traditional herbal medicines have been utilized for human and animal health for millenniums. Currently, phytotherapies are investigated as alternative methods for controlling coccidian infections. A number of herbal extracts have been proven to be efficient to control coccidiosis. Metwaly et al. (2012) reported the effect of Khodary date fruit extract from Phoenix dactylifera on the coccidiosis caused by Eimeria papillata. The date fruit extract can decrease the fecal oocyst output and reduce the parasites in the intestinal villi and associated histopathological lesions. It was evaluated that curcumin (diferuloylmethane), which is extracted from the perennial herb turmeric (Curcuma longa) can inhibit Eimeria tenella sporozoites viability and infectivity in vitro (Khalafalla et al. 2011). del Cacho et al. (2010) showed that artemisinin extracted from the natural herb Artemisia annua can alter the oocyst wall formation, reduce the sporulation rate, and inhibit expression of sarcoplasmicendoplasmic reticulum calcium ATPase in macrogamete stage. De Pablos et al. (2010) studied the anticoccidial efficacy of maslinic acid. The results showed the anticoccidial index (ACI) for maslinic acid was 210.27 compared 173.09 for control treatment. Maslinic acid can decrease lesions and oocyst counts and increase the body weight. Naidoo et al. (2008) demonstrated that three plant extracts, Tulbaghia violacea, Vitis vinifera, and Artemisia afra, can improve the feed conversion ratios of Eimeriainfected chickens in comparison to the untreated control group and T. violacea significantly decreases oocyst excretion. Michels et al. (2011) also proved that infected chickens when treated with coumestans from Eclipta alba displayed higher weight gains, improved feed efficiency and significantly

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decreased fecal oocysts and macroscopic lesions. Youn and Noh (2001) screened 15 different Asian herbal extracts for activity against E. tenella. Extract from Sophora flavescens Aiton was the most effective preparation as indicated by survival rates, diarrhea scores and lesion scores, body weight, and oocyst excretion. Many other plant extracts, such as tannins, xanthohumol, flaxseed oil, and betaine, have been screened to be effective in controlling Eimeria infection in vitro or in vivo (Molan et al. 2009; Allen et al. 1996, 1997; Allen 2007; Augustine et al. 1997; Augustine and Danforth 1999). Halofuginone hydrobromide is a synthesized analog of febrifugine, a constituent of the leaf and root of Chinese medicinal herb Dichroa febrifuga Lour. (Chinese name Chng Shn), which is an anticoccidial drug approved by the European Union and the US Food and Drug Administration Agency (Zhang et al. 2012a). Therefore we have evaluated the anticoccidial effects of a chemical extract of this herb as supplement in the broiler feed.

500 ml chloroform at room temperature for 1 day. The mixtures were heated with reflux of chloroform for 2 h. After filtration, the residues were suspended in 500 ml chloroform and extracted. The combined solvent was evaporated by heating. The extracted residue was grounded and stored in sealable plastic bag at 4 C until use. Experimental design A total of one hundred 12-day-old birds were weighed and randomly distributed into five groups of 20 birds. Birds in groups 1 and 2 were provided with 20 or 40 mg extract in 1 kg feed from day 12 to day 21. Group 3 was treated with 2 mg diclazuril/kg feed from day 14 to day 21, while group 4 and group 5 served as untreated controls throughout the experiment. On day 14, birds in groups 1, 2, 3, and 4 were orally infected with 1.5104 sporulated E. tenella oocysts. The clinical signs were observed and recorded each day until the end of the experiment. Evaluation of anticoccidial effect

Materials and methods Birds and parasites One-day-old male broilers were bought from a local hatchery in Hangzhou, People's Republic of China. The birds were reared in clean wire cages in hygienically controlled coccidian-free environment and provided with coccidiostatfree feed and water without any antibiotics. All procedures related to the animals and their care met the international guiding principles for biomedical research involving animals, as issued by the Council for the International Organizations of Medical Sciences (1985) and the World Association for the Advancement of Veterinary Parasitology guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys as previously mentioned (Holdsworth et al. 2004) along with the Guidelines for Keeping Experimental Animals issued by the government of People's Republic of China. The sporulated oocysts of E. tenella Shanghai strain were kindly gifted by Dr. Bing Huang, Shanghai Veterinary Research Institute, China Academy of Agricultural Sciences. The oocysts were stored in 2.5 % potassium dichromate reagent at 4 C and propagated in 2-week-old chickens before use. Preparation of extract According to the properties and previous described methods (Jiang et al. 2005; Murata et al. 1998), a modified chloroform reflux method was used to extract febrifugine. Briefly, 100 g dry powder of D. febrifuga root was macerated in

ACIACI relative ratio of body weight gains survival rate oocyst value median lesion scores was calculated to evaluate the anticoccidial efficacy (Johnson and Reid 1970; Ma et al. 2011). On the seventh day post-infection, the survival rate (SR) of each group was calculated and birds were weighed to determine the percentage increase in body weight gains (piBWG) and relative ratio of body weight gains (rBWG). SR was investigated as the number of surviving chickens/the number of initial chickens. The piBWG was calculated as piBWG body weight after infection body weight before infection = body weight before infection 100% . The rBWG was calculated for each group according to the following equation: rBWG piBWG in each group= piBWG in group of noninfected -unmedicated controls 100%. After being weighed, all surviving birds were subsequently euthanized. The overall general appearances of cecal lesions were recorded by two skilled persons who were not informed on the group allocation of the respective birds. Lesion scores were assessed according to the method of Johnson and Reid (1970). Generally, cecal lesions were ranked from 0 (normal) to 4 (dead or extremely severe) based on the macroscopical appearance of the intestine. Oocysts in pooled caecum contents of each bird were counted using a hemocytometer counting technique and the result was expressed as oocysts per gram (OPG) (9, 11). Oocyst value OPG in each group=OPG in group of infected-unmedicated controls 100. Protection rate (in percent) was calculated as OPG from infected- unmedicated control birds OPG from medicated birds = OPG from

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infected-unmedicated control birds 100% (Du and Wang 2005; Rose and Mockett 1983). Diarrhea scores were recorded between day 4 and day 7 post-infection according to Du and Hu (2004). Briefly, the percentage of samples containing visible blood was estimated for each group and scored as follows: 0, no hemorrhagic samples; 1, 25 % of the samples are hemorrhagic; 2, 50 % of the samples are hemorrhagic; 3, 75 % of the samples are hemorrhagic; and 4, all the samples are haemorrhagic. Statistical analyses The data were analyzed using the software SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). The parameters of body weight gains and OPG were analyzed by using one-way analysis of variance and mean values were compared using the Duncan's multiple range tests. The results were expressed as mean SD. The lesion scores and bloody diarrhea were statistically compared using KruskalWallis H test and the results were expressed as median (inter-quartile range). Statistical difference was accepted at the level of P <0.05.

Results Bloody diarrhea All birds survived throughout the experiment. All infected birds showed clinical symptom of coccidiosis, such as depression, somnolence, and feather disorders. Bloody feces were found in all infected groups but not in the uninfected birds. The most severe bloody diarrhea was observed in the infected-unmedicated group 4 and scores were significantly higher than in any of the other groups (P <0.05). Significantly fewer cases of bloody feces were recorded in infectedmedicated birds of groups 1 to 3 (P <0.05, Fig. 1). Lesion scores The surviving birds were euthanized at the end of the experiment and the lesion scores were evaluated immediately. As presented in Fig. 2, the most severe cecal damage was found in the infected-unmedicated group 4. The lesion scores in group 1 were significantly lower than in the infected-unmedicated control group 4 (P <0.05). Application of 40 mg extract/kg feed (group 2) reduces the lesion score but no statistical difference was found compared to the infected-untreated controls (P >0.05). Oocyst counts As shown in Table 1, treatment with the extract reduced oocyst counts. The OPG of groups 1, 2, and 3 was significantly lower

Fig. 1 Bloody diarrhea (median, IQR) of each group during the fourth and seventh day post-inoculation with E. tenella. Chickens in groups 1, 2, and 3 were infected and treated with 20, 40 mg extract/kg feed or 2 mg diclazuril/kg feed, respectively. Chickens in group 4 served as infected-unmedicated controls and group 5 uninfected-unmedicated controls. Each day ranging from the fourth to seventh day postinfection, the total bloody diarrhea were counted and ranked from 0 to 4, which mean normal feces (ranked 0), 25 %(ranked 1), 50 %(ranked 2), and 75 %(ranked 3) bloody feces of all the feces and absolutely complete bloody diarrhea(ranked 4), respectively. Columns with different letters present statistical difference (P <0.05)

than that in infected-unmedicated group (P <0.05). Birds treated with 40 mg/kg extract excreted statistically significant less oocysts than group 1 chicken (P <0.05) and the protection rate was higher than in group 1.

Fig. 2 Lesion scores (median, IQR) of caecum examined at the seventh day post-infection with E. tenella. Chickens in groups 1, 2, and 3 were infected and treated with 20, 40 mg extract/kg feed or 2 mg diclazuril/kg feed, respectively. Chickens in group 4 served as infectedunmedicated controls and group 5 uninfected-unmedicated controls. The lesion scores were ranked from 0 (absence of lesions) to 4 (extremely severe lesions and dead chickens due to coccidiosis). Columns with different letters mean statistical difference (P <0.05)

2232 Table 1 Average oocysts counts per gram of cecal content in each group and protection rate (in percent) Groups 1 2 3 4 5 (20 mg/kg) (40 mg/kg) (diclazuril) (infected-unmedicated) (noninfected-unmedicated) OPG (105) 56.290.20 b 42.450.27 c 8. 420.21 d 90.220.11 a 0.000.00 e Protection rate (%) 37.58 52.88 90.69 0.00 100.00

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The complete cecum contents of each bird were pooled separately and oocysts were counted from 1 g of the feces (oocysts per gram, OPG) using hemocytometer counting technique. Protection rate (%) was calculated as follows: Protection rate% the number of oocysts from infected unmedicated control birds the number of oocysts from medicated birds=the number of oocysts from infected unmedi cated control birds 100% . Columns with different letters mean statistically different (P <0.05)

Fig. 4 Anticoccidial index (ACI) of each group. Birds in groups 14 were infected with oocysts. Birds in groups 1, 2, and 3 were treated with plant extract 20, 40 mg/kg feed or 2 mg diclazuril/kg feed, respectively. Chickens in groups 4 and 5 served as infectedunmedicated and uninfected-unmedicated controls, respectively

Body weight The body weights of all groups are presented in Fig. 3. The BWG in group 4 (infected-unmedicated group) was significantly lower than that in groups 1, 3, and 5 (P <0.05). The BWG of birds treated with 20 mg extract/kg feed was significant higher than in infected-unmedicated control group (group 4) (P <0.05). ACI The ACI of all groups were described in Fig. 4. Chickens in the infected-unmedicated control group are shown with the lowest ACI value compared with the medicated groups and uninfected-unmedicated control group 5. A higher ACI was seen in group 1 (20 mg extract/kg feed) than in group 2 (40 mg extract/kg feed).

Discussion Chinese traditional herb D. febrifuga has been used against malarial fever for millenniums in China but little is known about its anticoccidial activity (Pines et al. 2000). In this study, the anticoccidial efficacy of D. febrifuga extract was investigated. Briefly, 20 mg extract/kg feed can reduce bloody diarrhea, intestinal lesions, and oocysts excretion and increase BWG. On the basis of the results of this study, D. febrifuga extract was effective as an anticoccidial to protect chickens against coccidiosis infection and was recommended to be used as a coccidiostat drug, and some further studies are still needed. Several compounds were isolated from the roots or leaves of D. febrifuga but the most active alkaloid against malarial fever was febrifugine, which is the main constituent of the plant extract (Pines et al. 2000). It is not determined in this study if the main anticoccidial content is febrifugine. This compound is not well known; however, it appears to share properties with halofuginone. Febrifugine is known to be relatively toxic to mammals and birds and thus the lower benefit of the higher dose observed in this study may be related to such adverse activities (Pines et al. 2000). Lesion scores and oocyst counts are commonly applied parameters to evaluate the anticoccidial efficacy of drugs. It was noted that OPG of group 2 were lower than in group 1, although other parameters of efficacy displayed contrasting effects. Similar results have been published by McKenzie et al. (1989). It appears that the relationship between oocyst counts and lesion scores is not congruent because oocyst

Fig. 3 Body weight (meanSD) before and after infection with E. tenella oocysts. Birds in groups 14 were infected with E. tenella sporulated oocysts. Birds in groups 1, 2, and 3 were treated with plant extract 20, 40 mg/kg feed or 2 mg diclazuril/kg feed, respectively. Chickens in groups 4 and 5 served as infected-unmedicated and uninfected-unmedicated controls, respectively. Columns with different letters mean statistical difference (P <0.05)

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2233 (WAAVP) guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys. Vet Parasitol 121(34):189212 Jiang S, Zeng Q, Gettayacamin M, Tungtaeng A, Wannaying S, Lim A, Hansukjariya P, Okunji CO, Zhu S, Fang D (2005) Antimalarial activities and therapeutic properties of febrifugine analogs. Antimicrob Agents Chemother 49(3):11691176 Johnson J, Reid WM (1970) Anticoccidial drugslesion scoring techniques in battery and floor-pen experiments with chickens. Exp Parasitol 28(1):3036 Khalafalla RE, Muller U, Shahiduzzaman M, Dyachenko V, Desouky AY, Alber G, Daugschies A (2011) Effects of curcumin (diferuloylmethane) on Eimeria tenella sporozoites in vitro. Parasitol Res 108(4):879886 Lai L, Bumstead J, Liu Y, Garnett J, Campanero-Rhodes MA, Blake DP, Palma AS, Chai W, Ferguson DJ, Simpson P, Feizi T, Tomley FM, Matthews S (2011) The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella. PLoS Pathog 7(10):e1002296 Ma D, Ma C, Pan L, Li G, Yang J, Hong J, Cai H (2011) Vaccination of chickens with DNA vaccine encoding Eimeria acervulina 3-1E and chicken IL-15 offers protection against homologous challenge. Exp Parasitol 127:208214 McKenzie ME, Conway DP, Dayton AD (1989) Predictive relationship of lesion scores to the number of Eimeria maxima oocysts inoculated in medicated and nonmedicated broilers. Poultry Sci 68 (suppl 1):92 Metwaly MS, Dkhil MA, Al-Quraishy S (2012) The potential role of Phoenix dactylifera on Eimeria papillata-induced infection in mice. Parasitol Res 111:681687 Michels MG, Bertolini LC, Esteves AF, Moreira P, Franca SC (2011) Anticoccidial effects of coumestans from Eclipta alba for sustainable control of Eimeria tenella parasitosis in poultry production. Vet Parasitol 177(12):5560 Molan AL, Liu Z, De S (2009) Effect of pine bark (Pinus radiata) extracts on sporulation of coccidian oocysts. Folia Parasitol (Praha) 56(1):15 Murata K, Takano F, Fushiya S, Oshima Y (1998) Enhancement of NO production in activated macrophages in vivo by an antimalarial crude drug, Dichroa febrifuga. J Nat Prod 61(6):729733 Naidoo V, McGaw LJ, Bisschop SP, Duncan N, Eloff JN (2008) The value of plant extracts with antioxidant activity in attenuating coccidiosis in broiler chickens. Vet Parasitol 153(34):214219 Pines M, Vlodavsky I, Nagler A (2000) Halofuginone: from veterinary use to human therapy. Drug Development Research 50:371378 Rose ME, Mockett AP (1983) Antibodies to Coccidia: detection by the enzyme-linked immunosorbent assay (ELISA). Parasite Immunol 5:479489 Williams RB (1999) A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry. Int J Parasitol 29(8):12091229 Youn HJ, Noh JW (2001) Screening of the anticoccidial effects of herb extracts against Eimeria tenella. Vet Parasitol 96(4):257263 Zhang DF, Sun BB, Yue YY, Yu HJ, Zhang HL, Zhou QJ, Du AF (2012a) Anticoccidial effect of halofuginone hydrobromide against Eimeria tenella with associated histology. Parasitol Res. doi:10.1007/s00436-012-2889-7 Zhang DF, Xu H, Sun BB, Li JQ, Zhou QJ, Zhang HL, Du AF (2012b) Adjuvant effect of ginsenoside-based nanoparticles (ginsomes) on the recombinant vaccine against Eimeria tenella in chickens. Parasitol Res. doi:10.1007/s00436-011-2784-7

counts increase faster than the severity of lesions (McKenzie et al. 1989; Holdsworth et al. 2004). It is suggested that the lesion scores should be assigned by only one judge who knows very well of the relationship between lesions and respective score rank, but knows nothing about the treatment of each bird.
Acknowledgments This work was supported by Wens Research Fund and Jiaxing Bureau of Science and Technology, Zhejiang Province, People's Republic of China (grant no. 2007BY6007). The authors thank the team of Veterinary Parasites' Pathology and Biology Laboratory, College of Animal Sciences, Zhejiang University.

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