Development And Application Of A Nested-PCR Assay For Detection Of Schistosoma Japonicum Infection In Domestic Animals

Schistosomiasis,a serious parasitic zoonosis,is still a public health problem in 76 countries and territories,where approximately 200 million people are symptomatically infected,and 20 million people are heavily infected with different parasite species of the genus Schistosoma.In China,the infected domestic animals,particularly buffaloes,cattle,goats and sheep,are the primary infection sources and play a vital role in disease transmission.The miracidium hatching test(MHT)using feces and antibody detection methods are the most common tests used in China for the identification of Schistosoma japonicum(S.Japonicum)infection in domestic animals.Because of the comprehensive control strategy implemented in China,the prevalence and intensity of infection in domestic animals with S.japonicum decreased to low levels.Therefore,the sensitivity and accuracy of MHT are low in this situation because of the low prevalence and infection rates.Antibody detection methods such as IHA have a high sensitivity but cannot discern active from previous infection and easily cross-react with antibodies from other parasites(parasitic flukes or helminths).Therefore,the development of highly sensitive diagnostics is essential.In recent years,various studies have shown that polymerase chain reaction(PCR)-based methods could be used to detect schistosome infection in humans and experimental animals and presented a high sensitivity and specificity.The present study aimed to develop a PCR-based method for detection of S.japonicum infection in domestic animals.Based on the previous published results in humans and experimental animals,the retrotransposon SjR2 was selected as expected PCR product from mitochondrial cytochrome c oxidase subunit ?(CO ?),mitochondrion coding region and the highly repetitive retrotransposon SjR2 in G55 A of S.japonicum by amplification from serum samples of artificially infected New Zealand rabbits.A specific nested-PCR assay was developed to detect S.japonicum infection in domestic animals via amplification of a 231-bp DNA fragment of retrotransposon SjR2.The specificity and sensitivity of the nested-PCR assay were determined by amplification of the expected product from the genomic DNA from worms of S.japonicum,Fasciola and Haemonchus contortus(H.Contortus),and a series of 1,2,5,10,20,40,and 80 S.japonicum eggs.In order to compare the nested-PCR results using serum and dry blood filter paper(DBFP)and to test the diagnostic possibility during the different stages of infection,the developed assay was first used in sera and DBFPs from goats and buffaloes at different time points of infection.Then,78 DBFPs from 39 artificially-infected bovines at 14 and 28 days post-infection and42 DBFPs from schistosome-negative bovines from the city of Huangshan in the Anhui province were used to evaluate the diagnostic validity.Furthermore,this assay was used to detect S.japonicum infection in domestic animals in Dongzhi and Wangjiang counties.The expected PCR product was detected in eggs and adult worms of S.japonicum and blood samples from S.japonicum-infected goats and water buffaloes,but not from Fasciola and H.contortus worms.This means that the nested-PCR assay has good specificity.The nested-PCR assay could detect the target S.japonicum DNA in the genomic DNA from only 1 egg.Both serum and DBFP could beused for diagnosis of S.japonicum infection in domestic animals and DBFP was better than serum.The nested-PCR assay in DBFP could amplify the expected product at days 3 and 4 post-infection in both goats and buffaloes and days 34 and 37 after treatment with praziquantel in goats,but not in sera.The sensitivity in buffaloes at 14 and 28 days post-infection was 92.30%(36/39)and 100%(39/39),respectively.The specificity was 97.60%(41/42).The positivity rates in Dongzhi and Wangjiang counties were 6.00% and 8.00% in bovines and 22.00% and 16.67% in goats,respectively.The positivity rates in goats in both counties were higher than those in bovines with a significant difference in Dongzhi county but not in Wangjiang county(P < 0.05 and P = 0.23,respectively).Our results suggest that the developed nested-PCR assay may be used for the diagnosis of S.japonicum infection in domestic animals,and the control of S.japonicum infection in goats should be paid more attention.