Development Of A QPCR For Detection Of Eimeria Spp.and Anticoccidial Effect Assessment Of Traditional Chinese Medicine

Ovine coccidia is parasitic protozoa which are belonging to Eimeriidae in the intestines of sheep and goats.After infection,lambs will have clinical symptoms such as diarrhea,bloody stool,decreased digestion and loss of appetite,resulting in the decline of lamb growth performance and even death.These parasitic diseases caused by Eimeria spp.are collectively called ovine coccidiosis.Ovine coccidiosis,as one of the most common parasitic diseases in sheep,seriously affects the health of lambs.There are many different species of Eimeria spp.,all of which can infect lambs.Several species mixed infections are common in clinic.In order to better prevent and control coccidiosis,a simple,rapid,high-throughput and high-sensitivity fluorescence quantitative PCR method was established for quantitative detection of sheep coccidia and to provide technical means for screening effective traditional Chinese medicine against coccidiosis.Artemisia annua extract,Coptis extract,Changshan extract,Quqiujing extract,Artemisia annua and pulsatilla were selected to feed lambs naturally infected with coccidia.The anticoccidial effect were evaluated from the changes of coccidial infection quantity and the effect on lamb growth performance,serum immune factors and intestinal microflora.Finally,Changshan extract and Artemisia annua were proved to have good anticoccidial effect,which provides a basis for the prevention and control of coccidiosis by traditional Chinese medicine instead of chemical drugs.1.Development of a qPCR for detection of Eimeria spp.In order to establish a SYBR Green real-time fluorescence quantitative PCR detection method,the specific primer was designed and synthesized based on the corresponding gene sequences of the Gen Bank,the reaction conditions and reaction system were optimized repectively,a rapid detection method on Eimeria spp.in ovis,SYBR Green real-time fluorescent quantitative PCR was established.The sensitivity,specificity and repeatability of the method are verified.The results showed that this method could effectively amplify the standard plasmid of Eimeria spp.gene with the concentration of 34-3.4×108 copies /μL.The standard curve showed a good linear relationship,and the detection sensitivity was 34 copies /μL.There was no cross reaction to Blastocystis,Microsporidia,Cryptosporidium,Giardia duodenum and other intestinal pathogens.The repeatability test showed that the coefficient of variation(CV)was less than 2%.This indicated that this method has good sensitivity,specificity and reproducibility,which is suitable for quantitative detection of a large number of samples and provides technical support for screening and effect evaluation of anticoccidial drugs.2.Screening on the traditional Chinese medicine which can be used to treat lamb coccidiosisthere are few studies on anti-coccidiosis traditional Chinese medicine for sheep compared with chickens and rabbits.In order to find suitable traditional Chinese medicines for the treatment of sheep coccidiosis,lambs naturally infected with coccidiosis were randomly divided into 8 group,which are control group,diclazuril group,Artemisia annua extract group,Coptis chinensis extract group,Changshan extract group,Artemisia annua group,pulsatilla group and Quqiujing extract group respectively.The constructed fluorescence quantitative PCR method was used to detect the infection intensity of coccidia in lambs before and after treatment.According to the anti-coccidiosis effect,the anti-coccidiosis traditional Chinese medicine was preliminarily screened,and then the effects of the selected drugs on lamb growth performance,serum immune factors and intestinal flora were evaluated to determine whether they can be used as drugs for the treatment of coccidiosis.The results showed that Changshan extract of 3g/d/each lamb could significantly reduce the infection amount of coccidia after continuous feeding for 1 week,and Artemisia annua fed with 20g/d/each lamb for 2 weeks could significantly reduce the infection amount of coccidia,and the efficacy of both were better than diclazuril,and it could still restrain coccidia after stopping treatment for 2 months and keep lambs at a low infection level(the average OPG of Changshan extract group,Artemisia group and diclazuril group were 2300,4850 and 15150 respectively after 2 months of treatment).Changshan extract and Artemisia annua could improve the daily weight gain of lambs.Compared with the control group,the relative weight gain rates of lambs in Changshan extract group and artemisia group were 140.13% and 119.08%respectively.During the 78-day experiment,the newly increased output value of each lamb in Changshan extract group and artemisia group was (?) 64.99 and (?) 40.54 respectively.Artemisia annua can increase the content of serum Ig G and enhance the immunity of lambs.Both Changshan extract and Artemisia annua could increase the diversity of intestinal flora and the abundance of Firmicutes in lambs.Changshan extract can increase the abundance of beneficial bacteria in the intestinal tract of lambs,which may be related to the increase of daily gain of lambs.In this study,Changshan extract and Artemisia annua were successfully screened as the effective traditional Chinese medicine for the treatment of lamb coccidiosis.