Prevalence Of Some Farms Of Isospora Suis In Fujian Province And Establishment Of Its PCR And LAMP Detection Methods

Isospora suis mainly inhabiting the jejunum and ileum of pigs,which can cause diarrhea and reduced weight gain of piglets within three weeks of age and lead to the economic losses.The objective of the present investigation was to determine the prevalence of Isospora suis in piglets in Fujian Province.2173 faecal samples of piglets from 1 week to 8 week were collected from 29 commercial swine farms in 9 areas of Fujian Province.The results showed that 28 pig farms have detected oocysts of I.suis,212(9.76%)piglet facecal samples were infected with Lsuis in these samples.The highest infection rate of Lsuis on each swine farms were 27.78%,and OPG values(Number of oocysts per gram of faeces)were from 25 to 115100.The positive rate of oocyst and the logarithm of OPG values were significantly correlated(R=0.445,P=0.016).The I.suis infection rate of piglet from week 1 to week 8 was 2.66%～17.46%.OPG values of 2-week-old piglets was higher than that of other week-old piglets significantly(P<0.01),and positive rate of 3-week-old piglets was than that of other week-old piglets significantly(P<0.01).The results showed that the infection rate and OPG values of Lsuis in the farms without using any anticocidial drugs treatment were significantly higher than that in those farms with using Toltrazuril(P<0.01,P<0.05).It was found that Positive rate and OPG values of Lsuis in fermentation bed were significantly higher than slatted floor(P<0.01,P<0.05).Seen from the survey,Lsuis is wide prevalence in Fujian Province,piglets treated with Toltrazuril can effectively control piglet coccidiosis,reduce the occurrence of piglet coccidiosis.As the existence of false and leak detection in traditional microscopic detection,the detection methods of molecular biology were studied in this test.According to the biological characteristics of Lsuis oocysts,we successfully extracted DNA from Lsuis oocysts by the methods of repeated freezing thawing and kit(containing lysozyme and proteinase K).This method can quickly break the wall of oocysts.Conditions of PCR reaction were optimized,the results showed,55.3℃ was the optimal annealing temperature,optimum concentration for template DNA and each primer(upstream and downstream)was 1.5μL,0.75μM,respectively.After the test to the sensitivity of primers,it was found that the lowest detectable amount of recombinant plasmid was 3.37x 104 Copies/μL,converted into concentration of plasmid DNA was 123fg/μL,extracting DNA from a certain number of oocysts for PCR amplification,the detection limit was 13 oocysts.To found a more sensitive detection method,real-time fluorescence detection method of LAMP was established according to 18S rRNA gene entire sequences of Lsuis by real-time PCR.The reaction conditions of this method were optimized.The results show that the optimum concentration for the Bst DNA polymerase,Mg2+,dNTPs,betaine,primer was 8U/25μL,8 mmol/L,1.6 mmol/L,1 mol/L,2μM,respectively.Optimal concentration ratio of outer and inner primers was10:1.In the sensitivity test of LAMP,the detection limit of the recombinant plasmid was 2.74×102 Copies/μL,converted into concentration of plasmid DNA was 1fg/μL.We can see the sensitivity of LAMP technology was about 100 times higher than PCR technology,extracting DNA from a certain number of oocysts for LAMP,the detection limit was 5 oocysts.