Molecular Identification Of Ba Ylisascaris Schroeder1from Qinl1ng Subspecies Of Giant Panda In China By The Nuclear Ribosomal DNA

Ascarid is the most common parasitic nematodes of giant panda, causing serious harm.In the present study, a total of20nematode isolates (including10male and10female worms)representing Baylisascaris schroederi from5Qinling subspecies of captive giant pandas(Ailuropoda melanoleuca) captured in Qinling mountain and raised in Shaanxi Rare WildlifeRescue Breeding Research Center in Shaanxi province of China, were characterized andgrouped genetically by the internal transcribed spacer (ITS),5.8S,18S,28S and IGS ofnuclear ribosomal DNA (rDNA). Based on these sequences, the sequence variability wereexamined within B. schroederi and among parasites in order Ascaridata available inGenBank, and phylogenetic relationships among these species in the Ascarididae werere-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximumlikelihood (ML) analyses. The results were as follows:In the first part, the rDNA fragment spanning3’ end of18S rDNA, complete ITS-1rDNA, and5’ end of5.8S rDNA was amplified and sequenced. The sequences of ITS-1rDNA for all the B. schroederi isolates were427bp in length, with no genetic variationdetected among these isolates. Phylogenetic analyses based on the ITS-1rDNA sequencesrevealed that all the male and female B. schroederi isolates sequenced in the present studywere posited into clade of genus Baylisascaris, sistered to zoonotic nematodes in genusAscaris, and the ITS-1rDNA sequence could distinguish different species in order Ascaridata.These results showed that the ITS-1rDNA provided a suitable molecular marker for theinter-species phylogenetic analysis and differential identification of nematodes in orderAscaridata; The lengths of B. schroederi5.8S and ITS-2rDNA sequences were156bp and327bp, respectively, and no nucleotide variation was found in these two rDNA regions amongthe20B. schroederi samples examined, and these ITS-2sequences were identical to that of B.schroederi isolated from giant panda in Sichuan province, China. The interspecies differencesin5.8S and ITS-2rDNA sequences among members of the family Ascarididae were0–1.3%and0–17.7%, respectively. All B. schroederi samples clustered together and sistered to B.transfuga with high posterior probabilities/bootstrap values, which further confirmed thatnematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequencepositions (difficulty of inferring homology by positions), ITS-2sequence alone is likelyunsuitable for phylogenetic analyses at the family level, but the combined5.8S and ITS-2rDNA sequences provide alternative genetic markers for the identification of B. schroederiand for phylogenetic analysis of parasites in the family Ascarididae.The second part was to sequence and analyse the18S rDNA and28S rDNA. The lengthof18S was630bp, the20samples showed a similarity of100%; the length of28S was476bp, with the sequence differences varied between0.2%and0.6%. Then, the phygenetic treewas re-constructed based on the combined sequences of18S and28S using series of methods.It was difficult to identify the B. schroederi from the other parasites in genus Baylisascaris,indicating that the18S and28S were not suitable to be the genetic markers for identificationof species in genus Baylisascaris.In the last part, we sequenced and analyse the intergenetic spacer sequence of B.schroederi. The organization of IGS rDNA sequences was similar to that of other eukaryotes.The length of IGS was approximately427bp, the analysis of twenty samples was showed thatsequence differences varied from0to6.4%between species. All these sequences containedonly inverted repeats. The results suggested that the gene IGS of B. schroederi had a highhomology and intraspecific identification can not distinguish the isolates. The marker may beused to identify the high level as a potential marker.The present study examined the genetic variations and population structure of B.schroederi based on the nuclear ribosomal DNA. The results were able to reflect the geneticvariability and population structure of B. schroederi in mainland China, and provided usefulmolecular information for the prevention and control to diseases of giant panda.