Parasitol Res (2012) 111:2229–2233 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 evalu- use of anticoccidial drugs (Hao et al. 2007). The domestic ate the anticoccidial efficacy of an extract of Chinese traditional poultry industry of People's Republic of China primarily relies herb Dichroa febrifuga Lour. One hundred broiler birds were on medical prophylaxis. But the emergence of problems re- assigned to five equal groups. All birds in groups 1–4were lated to drug resistance and drug residues of antibiotics in the orally infected with 1.5×104 Eimeira tenella sporulated chicken meat has stimulated us to seek safer and more effica- oocysts and birds in groups 1, 2 and 3 were medicated with cious alternative control strategies (Lai et al. 2011). 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respec- Chinese traditional herbal medicines have been utilized for tively. The bloody diarrhea, oocyst counts, intestinal lesion human and animal health for millenniums. Currently, phyto- scores, and the body weight were recorded to evaluate the therapies are investigated as alternative methods for control- anticoccidial efficacy. The results showed that D. febrifuga ling coccidian infections. A number of herbal extracts have extract was effective against Eimeria infection; especially been proven to be efficient to control coccidiosis. Metwaly et 20 mg D. febrifuga extract/kg feed can significantly increase al. (2012) reported the effect of Khodary date fruit extract body weight gains and reduce bloody diarrhea, lesion score, from Phoenix dactylifera on the coccidiosis caused by Eime- and oocyst excretion in comparison to infected-unmedicated ria papillata. The date fruit extract can decrease the fecal control group. 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 Introduction perennial herb turmeric (Curcuma longa) can inhibit Eimeria tenella sporozoites viability and infectivity in vitro (Khalafalla Coccidiosis may cause severe intestinal diseases. Coccidiosis et al. 2011). del Cacho et al. (2010)showedthatartemisinin is distributed worldwide in poultry and wild birds and is of extracted from the natural herb Artemisia annua can alter the major economic impact in poultry production due to mortality oocyst wall formation, reduce the sporulation rate, and inhibit and morbidity. Epidemiologic studies have shown that the expression of sarcoplasmic–endoplasmic reticulum calcium mortality of fowl can range from 5 to 70 % (Du and Hu ATPase in macrogamete stage. De Pablos et al. (2010)studied 2004; Michels et al. 2011). Economic losses caused by avian the anticoccidial efficacy of maslinic acid. The results showed coccidiosis, including decreased productivity, usage of cocci- the anticoccidial index (ACI) for maslinic acid was 210.27 diostat, and vaccines, are estimated to amount to $3 billion compared 173.09 for control treatment. Maslinic acid can annually (Dalloul and Lillehoj 2006; Lai et al. 2011; Williams decrease lesions and oocyst counts and increase the body 1999; Zhang et al. 2012b; Michels et al. 2011). In China alone, weight. Naidoo et al. (2008) demonstrated that three plant the estimated expenditure is about $30–60 million due to the extracts, Tulbaghia violacea, Vitis vinifera, and Artemisia afra, can improve the feed conversion ratios of Eimeria- : : : : infected chickens in comparison to the untreated control group D.-F. Zhang B.-B. Sun Y.-Y. Yue Q.-J. Zhou A.-F. Du (*) and T. violacea significantly decreases oocyst excretion. Institute of Preventive Veterinary Medicine, Michels et al. (2011) also proved that infected chickens when College of Animal Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China treated with coumestans from Eclipta alba displayed higher e-mail: [email protected] weight gains, improved feed efficiency and significantly 2230 Parasitol Res (2012) 111:2229–2233 decreased fecal oocysts and macroscopic lesions. Youn and 500 ml chloroform at room temperature for 1 day. The Noh (2001) screened 15 different Asian herbal extracts for mixtures were heated with reflux of chloroform for 2 h. activity against E. tenella. Extract from Sophora flavescens After filtration, the residues were suspended in 500 ml chlo- Aiton was the most effective preparation as indicated by roform and extracted. The combined solvent was evaporated survival rates, diarrhea scores and lesion scores, body by heating. The extracted residue was grounded and stored weight, and oocyst excretion. Many other plant extracts, in sealable plastic bag at 4 °C until use. such as tannins, xanthohumol, flaxseed oil, and betaine, have been screened to be effective in controlling Eimeria Experimental design infection in vitro or in vivo (Molan et al. 2009; Allen et al. 1996, 1997; Allen 2007; Augustine et al. 1997;Augustine A total of one hundred 12-day-old birds were weighed and and Danforth 1999). randomly distributed into five groups of 20 birds. Birds in Halofuginone hydrobromide is a synthesized analog of groups 1 and 2 were provided with 20 or 40 mg extract in febrifugine, a constituent of the leaf and root of Chinese 1 kg feed from day 12 to day 21. Group 3 was treated with medicinal herb Dichroa febrifuga Lour. (Chinese name 2 mg diclazuril/kg feed from day 14 to day 21, while group Cháng Shān), which is an anticoccidial drug approved by 4 and group 5 served as untreated controls throughout the the European Union and the US Food and Drug Adminis- experiment. On day 14, birds in groups 1, 2, 3, and 4 were tration Agency (Zhang et al. 2012a). Therefore we have orally infected with 1.5×104 sporulated E. tenella oocysts. evaluated the anticoccidial effects of a chemical extract of The clinical signs were observed and recorded each day this herb as supplement in the broiler feed. until the end of the experiment.

Evaluation of anticoccidial effect Materials and methods ACI½ ACI ¼ ðrelative ratio of body weight gains þ survival Birds and parasites rateÞðÞoocyst value þ median lesion scores was calculat- ed to evaluate the anticoccidial efficacy (Johnson and Reid One-day-old male broilers were bought from a local hatchery 1970;Maetal.2011). in Hangzhou, People's Republic of China. The birds were On the seventh day post-infection, the survival rate (SR) reared in clean wire cages in hygienically controlled of each group was calculated and birds were weighed to coccidian-free environment and provided with coccidiostat- determine the percentage increase in body weight gains free feed and water without any antibiotics. All procedures (piBWG) and relative ratio of body weight gains (rBWG). related to the animals and their care met the international SR was investigated as the number of surviving chickens/the guiding principles for biomedical research involving animals, number of initial chickens. The piBWG was calculated as issued by the Council for the International Organizations of as piBWG ¼ ðbody weight after infection body weight Medical Sciences (1985) and the World Association for before infectionÞ = ðÞbody weight before infection 100%. the Advancement of Veterinary Parasitology guidelines The rBWG was calculated for each group according to for evaluating the efficacy of anticoccidial drugs in chick- the following equation: rBWG ¼ ðÞpiBWG in each group = ens and turkeys as previously mentioned (Holdsworth et al. ðpiBWG in group of noninfected-unmedicated controlsÞ 2004) along with the Guidelines for Keeping Experimental 100%. Animals issued by the government of People's Republic of After being weighed, all surviving birds were subse- China. quently euthanized. The overall general appearances of ce- The sporulated oocysts of E. tenella Shanghai strain were cal lesions were recorded by two skilled persons who were kindly gifted by Dr. Bing Huang, Shanghai Veterinary Re- not informed on the group allocation of the respective birds. search Institute, China Academy of Agricultural Sciences. Lesion scores were assessed according to the method of The oocysts were stored in 2.5 % potassium dichromate Johnson and Reid (1970). Generally, cecal lesions were reagent at 4 °C and propagated in 2-week-old chickens ranked from 0 (normal) to 4 (dead or extremely severe) before use. based on the macroscopical appearance of the intestine. Oocysts in pooled caecum contents of each bird were Preparation of extract counted using a hemocytometer counting technique and the result was expressed as oocysts per gram (OPG) (9, According to the properties and previous described methods 11). Oocyst value ¼ ðÞOPG in each group =ðOPG in group of (Jiang et al. 2005; Murata et al. 1998), a modified chloro- infected-unmedicated controlsÞ100. Protection rate (in form reflux method was used to extract febrifugine. Briefly, percent) was calculated asð OPG from infected-unmedicated 100 g dry powder of D. febrifuga root was macerated in control birds OPG from medicated birdsÞ = ðOPG from Parasitol Res (2012) 111:2229–2233 2231 infected-unmedicated control birdsÞ100% (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 estimat- ed 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 Fig. 1 Bloody diarrhea (median, IQR) of each group during the fourth the Duncan's multiple range tests. The results were expressed and seventh day post-inoculation with E. tenella. Chickens in groups 1, as mean ± SD. The lesion scores and bloody diarrhea were 2, and 3 were infected and treated with 20, 40 mg extract/kg feed or statistically compared using Kruskal–Wallis H test and the 2 mg diclazuril/kg feed, respectively. Chickens in group 4 served as infected-unmedicated controls and group 5 uninfected-unmedicated results were expressed as median (inter-quartile range). Sta- controls. Each day ranging from the fourth to seventh day post- tistical difference was accepted at the level of P<0.05. infection, 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. Col- Results umns with different letters present statistical difference (P<0.05)

Bloody diarrhea than that in infected-unmedicated group (P<0.05). Birds trea- ted with 40 mg/kg extract excreted statistically significant less All birds survived throughout the experiment. All infected oocysts than group 1 chicken (P<0.05) and the protection rate birds showed clinical symptom of coccidiosis, such as de- washigherthaningroup1. pression, 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). Signifi- cantly fewer cases of bloody feces were recorded in infected- medicatedbirdsofgroups1to3(P<0.05, Fig. 1).

Lesion scores

The surviving birds were euthanized at the end of the experiment and the lesion scores were evaluated immediate- ly. 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). Applica- tion of 40 mg extract/kg feed (group 2) reduces the lesion score but no statistical difference was found compared to the Fig. 2 Lesion scores (median, IQR) of caecum examined at the sev- infected-untreated controls (P>0.05). enth 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 Oocyst counts diclazuril/kg feed, respectively. Chickens in group 4 served as infected- unmedicated controls and group 5 uninfected-unmedicated controls. The lesion scores were ranked from 0 (absence of lesions) to 4 (ex- As shown in Table 1, treatment with the extract reduced oocyst tremely severe lesions and dead chickens due to coccidiosis). Columns counts. The OPG of groups 1, 2, and 3 was significantly lower with different letters mean statistical difference (P<0.05) 2232 Parasitol Res (2012) 111:2229–2233

Table 1 Average oocysts counts per gram of cecal content in each group and protection rate (in percent)

Groups OPG (×105) Protection rate (%)

1 (20 mg/kg) 56.29±0.20 b 37.58 2 (40 mg/kg) 42.45±0.27 c 52.88 3 (diclazuril) 8. 42±0.21 d 90.69 4 (infected-unmedicated) 90.22±0.11 a 0.00 5 (noninfected-unmedicated) 0.00±0.00 e 100.00

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 rateðÞ¼ % ðthe number of oocysts from infected unmedicated control birds the number of oocysts Fig. 4 Anticoccidial index (ACI) of each group. Birds in groups 1–4 from medicated birdsÞ=the number of oocysts from infected unmedi were infected with oocysts. Birds in groups 1, 2, and 3 were treated cated control birds 100%. Columns with different letters mean sta- with plant extract 20, 40 mg/kg feed or 2 mg diclazuril/kg feed, tistically different (P<0.05) respectively. Chickens in groups 4 and 5 served as infected- unmedicated and uninfected-unmedicated controls, respectively Body weight seen in group 1 (20 mg extract/kg feed) than in group 2 The body weights of all groups are presented in Fig. 3. The (40 mg extract/kg feed). BWG in group 4 (infected-unmedicated group) was signif- icantly lower than that in groups 1, 3, and 5 (P<0.05). The BWG of birds treated with 20 mg extract/kg feed was signif- Discussion icant higher than in infected-unmedicated control group (group 4) (P<0.05). Chinese traditional herb D. febrifuga hasbeenusedagainst malarial fever for millenniums in China but little is known ACI about its anticoccidial activity (Pines et al. 2000). In this study, the anticoccidial efficacy of D. febrifuga extract was investi- The ACI of all groups were described in Fig. 4. Chickens in gated. Briefly, 20 mg extract/kg feed can reduce bloody diar- the infected-unmedicated control group are shown with the rhea, intestinal lesions, and oocysts excretion and increase lowest ACI value compared with the medicated groups and BWG. On the basis of the results of this study, D. febrifuga uninfected-unmedicated control group 5. A higher ACI was 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 Fig. 3 Body weight (mean±SD) before and after infection with E. parameters to evaluate the anticoccidial efficacy of drugs. It tenella oocysts. Birds in groups 1–4 were infected with E. tenella was noted that OPG of group 2 were lower than in group 1, sporulated oocysts. Birds in groups 1, 2, and 3 were treated with plant although other parameters of efficacy displayed contrasting extract 20, 40 mg/kg feed or 2 mg diclazuril/kg feed, respectively. effects. Similar results have been published by McKenzie et Chickens in groups 4 and 5 served as infected-unmedicated and uninfected-unmedicated controls, respectively. Columns with different al. (1989). It appears that the relationship between oocyst letters mean statistical difference (P<0.05) counts and lesion scores is not congruent because oocyst Parasitol Res (2012) 111:2229–2233 2233 counts increase faster than the severity of lesions (McKenzie (WAAVP) guidelines for evaluating the efficacy of anticoccidial – – et al. 1989; Holdsworth et al. 2004). It is suggested that the drugs in chickens and turkeys. Vet Parasitol 121(3 4):189 212 Jiang S, Zeng Q, Gettayacamin M, Tungtaeng A, Wannaying S, Lim A, lesion scores should be assigned by only one judge who Hansukjariya P, Okunji CO, Zhu S, Fang D (2005) Antimalarial knows very well of the relationship between lesions and activities and therapeutic properties of febrifugine analogs. Anti- respective score rank, but knows nothing about the treatment microb Agents Chemother 49(3):1169–1176 — of each bird. Johnson J, Reid WM (1970) Anticoccidial drugs lesion scoring tech- niques in battery and floor-pen experiments with chickens. Exp Parasitol 28(1):30–36 Acknowledgments This work was supported by Wens Research Khalafalla RE, Muller U, Shahiduzzaman M, Dyachenko V, Fund and Jiaxing Bureau of Science and Technology, Zhejiang Prov- Desouky AY, Alber G, Daugschies A (2011) Effects of curcumin ince, People's Republic of China (grant no. 2007BY6007). The authors (diferuloylmethane) on Eimeria tenella sporozoites in vitro. 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