| Chicken coccidiosis is one of the most serious parasitic disease in poultry that causes heavy economic losses each year to the poultry industry.Currently,the prevention and treatment of coccidiosis rely primarily on anticoccidial drugs.However,the prolonged use of these drugs has given rise to emergence of resistant Eimeria strains,resulting in decreased drug efficacy or therapy failure.Therefore,new anticoccidial drugs are urgently needed.Medicinal plant Brucea javanica possess anti-cancer,anti-amoeba,anti-babesia and anti-malaria activities.However,whether it has anti-coccidial activity remains unknown.In this study,the influence of B.javanica extract on the proliferation and morphological changes of cultured coccidiawas evaluated and its therapeutic effect on coccidiosis was investigated.1.Preparation and chemical composition analysis of Brucea javanicaextractThin-layer chromatography and gas chromatography-mass spectrometry(GC-MS)analysis were used for the identification and analysis of the medicinal plant and B.javanica extracts were prepared by ethanol refluxing method combined with extraction method.Morphological observation and thin-layer chromatography confirmed that the Brucea javanica(L.)Merr.(Ya-Dan-Zi)was the dried mature fruits of the plant.Three kinds of B.javanica extracts were prepared,including B.javanica petroleum ether extract(BJ-PE),B.javanica ethonal extract(BJ-EE)and B.javanica water extract(BJ-WE).B.javanica oil was comprised of oleic acid and other longchain fatty acids,fatty alcohols and a small amount of aromatic volatile components.2.Therapeutic effect of the extract of Brucea javanica on chicken coccidiosisA series of experiments was conducted to investigate the prophylactic and therapeutic effects of ethanol extract from B.javanica on chicken coccidiosis.In experiment 1,chickens were randomly assigned to 5 groups with 10 birds per group.Chickens were infected with Eimeria tenella and treated with B.javanica extract at 2.5(Group 1)or5 g(Group 2)raw herbs/kilogram feed or with 3 mg halofuginone hydrobromide/kilogram feed.Group 4 and 5 were served as infected-unmedicated and uninfected-unmedicated controls.The degree of bloody stool,number of oocysts and tissue lesion were examined daily post infection.Infected birds treated with B.javania extracts showed reduced scores of bloody diarrhea and tissue lesion than the infected-unmedicated control.The extract had no significant effect on BWG.Additionally,the anti-coccidia index(ACI)value of B.javanica extract at the given doses was only 131,indicating a slight anticoccidial effect.Based on above results,another experiment(experiment 2)was conducted to test the therapeutic effect of B.javanica extract at a higher dosage that was equivalent to 10 g raw herbs per kilogram feed.The parameters detectedwere the same as in experiment 1.The ACI of the B.javanica extract at this level was increased to 146,suggesting a moderate anticoccidial effect.In experiment 3,the number of chickens in each group was expanded to 20.Supporting the results in both experiments 1 and 2.B.javanica extract supplementation reduced the incidenceof bloody stool,the number of coccidia oocysts output,the second generation schizonts in cecumas well as the score of cecumlesion(P<0.05).The ACI value were 154,161 and 155 in low,medium and high dose of B.javanica extract,respectively,indicative of a moderate-to-good anti-coccidiosis effect.Taken together,the results reported here suggest that Brucea javanica has therapeutic effect on chicken coccidiosis.3.In vitro anti-coccidial activity of Brucea javanica extract and brucitin-D The Eimeria tenella-infected MDBK cell model was used to investigate the anti-coccidial activity of B.javanica ethonal extract(BJ-EE)and Bruceine-D.The cytotoxic of BJ-EE and Bruceine-D on MDBK cells was determined by MTT assay.The cells exposed to BJ-EE(2 μg/ml)or Bruceine-D(0.8 μg/ml)alone for 65 h showed a survival rate above 90%.We therefore treated the cells with BJ-EE at 2,1,0.5,0.25 and 0.125 μg/ml)or Bruceine-D at 0.8,0.4,0.2,0.1 and 0.05 μg/ml)to evaluate their anti-coccidial activity.After 24 h,48 h and 65 h,the DNA replication ofEimeria tenella was detected by real time PCR.BJ-EE treatment resulted in a significant reduction in the DNA level of coccidia regardless of the time duration and dosages.Treatment with Bruceine-D,alsosignificantly inhibited coccidia proliferation,with an exception of Bruceine-D at 0.05 μg/ml for 48 h.In addition,Bruceine-D exposure inhibited coccidia proliferation in a dose-dependent manner at the time points of 48 h and 65 h.Taken together,our in vitro study suggests that BJ-EE and Bruceine-D has anti-coccidial activity.4.The underlying mechanisms of Brucea javanica against chicken coccidiosisIn order to understand the mechanisms by which B.javanica extracts exert effect on chicken coccidiosis,freshly obtained non-sporulated oocysts were exposed to different concentrations of petroleum ether extract(BJ-PE),ethanol extract(BJ-EE),water extract(BJ-WE)and Bruceine-D and the morphology of sporozoite was monitored under optical microscope and transmission electron microscopy(TEM).It was found that BJ-EE,BJ-WE and Bruceine-D inhibited the sporulation of oocysts.BJ-EE and Bruceine-D treated sporozoites became shrunk,rounded,shortened with obscured cell border,membranolysis,disordered arrangement of organelle and dumb-bell appearance.These results indicate that Brucea javanica has a mild effect on chicken coccidian oocyst sporulation and can kill the sporozoite.In summary,the present study prepared the petroleum ether extract(oil),ethanol extract and water extract of B.javanica.The oil of B.javanica mainly contained fatty acids such as oleic acid or fatty alcohol compounds.B.javanica ethanol extract showed anti-coccidiosis effect in artificial infected chicken coccidiosis model.It effectively control the occurrence of blood stool,decrease the number of oocysts output and reduce the number of the second generation schizont in chicken cecum.The extracts had no effect on the BWG of chickens.B.javanica ethanol extract and Bruceine-D inhibited the proliferation of coccidial in MDBK cells.The extract of B.javanica effectively killed sporozoite,while the mechanisms involved warrant further study.The study provides a new candidate for the development of drugs for prevention and treatment of coccidiosis in chickens. |
