Improvement Of The Fecal Detection Method And Establishment Of A Specific PCR For Isospora Suis

Swine coccidiosis,which shows a series of major symptoms such as diarrhea and emaciation,is caused by Eimeria spp.and Isospora suis that parasitize the intestinal epithelial cells of pigs.Although there are many species of coccidium reported worldwide,only I.suis and 10 species of the genus Eimeria infected in swine have been confirmed by morphology and specific PCR.Pigs in different varieties and growth stages can be susceptible to coccidium,of which piglets in lactation have the highest susceptibility to I.suis with a high clinical tendency for watery diarrhea,while others have an asymptomatic infection.I.suis is widely prevalent and causes high financial loss to the pig breeding industry.Therefore,accurate and effective detection of I.suis is urgently needed,but the conventional diagnostic methods have many problems,such as a low detection rate.This study aimed to explore a method for a high detection rate as well as easy and simple operation by improving traditional detection methods of swine coccidiosis and to establish a specific PCR based on 18 S r RNA gene sequences of I.suis.The traditional detection method for I.suis is saturated saline floatation.However,it turns out to be very difficult to conduct in Anthony pigs.The reason is mainly on that Anthony pigs have incomplete developed digestive system and weak body resistance,and they absorb nutrition entirely from b reast milk.Their digestive tracts can be easily damaged so that the intestinal tracts cannot completely absorb nutrition,such as fat and protein,which will cause unabsorbed nutrition in the feces.When the saturated saline floatation is applied to detect I.suis,the fat will float over the solution and the protein will salt out,which will influence the field of view during microscopic examination.Therefore,in this study,urea was used instead of saturated saline to develop a improved method for I.suis detection.Thespecific gravity of saturated urea solution is equal to that of saturated saline solution,and urea can cause protein denaturation.Soap grain(the main component of which is higher aliphatic acid)can be used to remove fat.Metasilicate defrother can solve the bubble problem.The improved fecal detection methodcould detect more occysts of I.suisthan the traditional detection methoddid in experiment that simulate clinical situation(nearly 20 times).The detected rates of clinical samples were 35%,25% and 33% respectively for 3 pig farms using the improved fecal detection method,but were 5%,5% and 10% using traditional detection method.As a result,the improved methoed using urea,soap grain and metasilicate defrother can effectively remove most of the fat and protein,so caneliminate the impact of fat and protein in feces,which clears the field of view during microscopic examination and increases the detection rate.In this study,a specific PCR was established to detect I.suis with high sensitivity,rapidity and specificity.The specific primers were designed based on 18 S r RNA gene sequences of I.suis.The best reaction temperature was 55.3°C.Sample DNA from controls were used in specificity tests,including Eimeria suis,Eimeria tenella,Isospora canis,Toxoplasma gondii,Streptococcus,Escherichia coli,Clostridium welchii and Cryptosporidium baileyi.I.suis could be amplified solely using a specific primer,while the sample DN A of other species and negative controls could not be amplified.The minimum detectable DN A with the specific primers of CTS-5F/CTS-5R was 3 oocysts of I.suis.The clinical fecal samples were examined for the presence of I.suis with saturated saline floatation,the improved detection method(soap grain and urea and metasilicate defrother)and the specific PCR method.The results showed the improved fecal detection method could overcome the defect of traditional method.The detection rate of I.suis could remarkably increase.Moreover,the specific PCR had a higher detection rate with better specificity and sensitivity,compared with the improved fecal detection method.Therefore,the specific PCR is much better than fecal detection methods for detection rate.The improvement in the detection rate for I.suis would be helpful for drug usage and prompt treatment.