Genetic Identification And Population Structure Of Enterocytozoon Bieneusi In Captive Giant Pandas

The giant panda is the national treasure of our country and belongs to rare and vulnerable wildlife.Intestinal parasitic disease is one of the common diseases of the giant pandas.There are at least 9 parasite species that are parasitic on the intestinal tract of the giant panda,including 4 protozoa species:Cryptosporidium giant panda,Cryptosporidium andersoni（C.andersoni）,Toxoplasma gondii（T.gondii）,and Enterocytozoon bieneusi（E.bieneusi）which is considered as an obligate intracellular eukaryotic pathogen having the ability to infect invertebrate and vertebrate including human,domestic animals and wildlife,causing diarrhea and wasting syndrome.We surveyed a total of 200 captive giant pandas in China for the prevalence of E.bieneusi by using PCR and sequence analysis of the ribosomal internal transcribed spacer（ITS）.The captive giant pandas were from Chengdu Research Base of Giant Panda Breeding（n=75）,Dujiangyan Giant Panda Base（n=30）,Wolong Gengda Giant Panda Base（n=37）,Wolong Hetaoping Giant Panda Base（n=10）,Yaan Bifengxia Giant Panda Base（n=17）,and 15 zoological gardens（n=31）.The result showed,69 captive giant pandas were infected E.bieneusi,with an infection rate of 34.5%.Differences between the tested conservation bases and zoological gardens in infection rates and the prevalences among different age groups and genders were not significant（P>0.05）.Twelve genotypes were identified with seven known genotypes（SC02,EpbC,CHB1,SC01,D,F,and Peru 6）and five novel genotypes（SC04,SC05,SC06,SC07,and SC08）.Phylogenetic analysis showed11 genotypes belong to zoonotic group 1,indicating a public threat.We amplified MS1,MS3,MS4,and MS7 loci of the 69 E.bieneusi isolates by nested PCR to analyze the multilocus genotypes（MLGs）.There were 48,45,50,and 47specimens were amplified successfully in MS1,MS3,MS4,and MS7 loci and formed 10,8,9,and 5 types,respectively,according to the multilocus genotype analysis.At all four loci,36 specimens were successfully amplified and sequenced,forming 24 MLGs.Sequence polymorphism analysis showed E.bieneusi in both microsatellites and minisatellite include single nucleotide polymorphisms（SNP）and length polymorphisms.The phylogenetic analysis of E.bieneusi based on microsatellite and minisatellite revealed that sequences from host-adapted ITS E.bieneusi genotypes form a cluster,while those from zoonotic ITS genotypes formed another cluster.We surveyed 69 specimens positive for E.bieneusi from captive giant pandas in China based on ITS,MS1,MS3,MS4,and MS7 loci to infer E.bieneusi population genetics.Multilocus sequence analysis revealed a strong and significant intragenic linkage disequilibrium（LD）with limited recombination,indicating a clonal population.This result was further supported by measurements of pairwise intergenic LD and a standardized index of association（I_A^S）from allelic profile data.Analysis in STUCTURE suggested three sub-populations in E.bieneusi were formed,and right’s fixation index(F_ST)further confirmed the presence of three sub-populations.Further analysis indicates that sub-populations 1 and 2 had epidemic structure,while sub-population 3 exhibited a clonal structure.In conclusion,the captive giant pandas in China have a relatively high infection rate（34.5%）,and are mainly infected with SC02 genotype,which have a zoonotic potential.The genetic diversity within the giant panda-derived E.bieneusi isolates is relatively high.Our study firstly analyzes the population genetics of E.bieneusi from captive giant pandas,revealing a clonal population.Our results provide a basis for the prevention and control of microporidiosis in giant pandas.