Identification Of Blastocystis Subtypes In Captive Wild Animals And Mechanism Research Of ST4 Colonization On Host Intestinal Response

Blastocystis is a common zoonotic intestinal parasite with controversial pathogenicity.To date,Blastocystis has been identified in domestic,companion animals and captive wildlife,and the subtype(ST)and genetic characteristics of Blastocystis are vary greatly.The effects of Blastocystis on the host’s intestinal microbiota and adaptive immune response lack systematic research as well.The present study firstly investigated the prevalence of Blastocystis in several captive wild animals in Sichuan province,and then further explored the subtype characteristics,host distribution,and genetic evolutionary relationship of Blastocystis in these hosts.Secondly,a specific subtype(ST4),one of the most common subtypes infecting humans and animals,was used in the present study to reveal its impacts on the host intestinal microbiota and adaptive immune response.Finally,by establishing an experimental colitis model,we investigated the role of ST4 in experimentally induced mouse colitis,and explored the possible mechanism.The main research contents of this thesis are as follows:Firstly,588 fecal samples of captive wild animals were collected in Sichuan province.The presence of Blastocystis was determined by amplifying the SSU r RNA gene of Blastocystis.Clustal X and MEGA 6 were used for clustering and genetic evolution analysis of Blastocystis-positive sequences.The results showed that the prevalence of Blastocystis in captive wild animals was 14.5%(85/588),and 9 different subtypes were found,including 6zoonotic subtypes(ST1-5 and ST8)and three host-specific subtypes(ST10,ST14 and ST17).Multilocus sequence typing(MLST)showed that different subtypes have genetic polymorphisms.Genetic evolution analysis further revealed that the subtypes identified in the present study were clustered in the same branch with human isolates,indicating that these subtypes have zoonotic potential.Secondly,by establishing an oral Blastocystis colonization mice model,we recorded the daily weight changes and disease activity index(DAI)of the mice,and conducted histopathological examination of the intestinal tissues of the mice.The mouse fecal samples were subjected to 16 S r RNA sequencing,and flow cytometry was used to detect the characteristic cytokines secreted by T helper cells(T helper,Th): Th1,Th2,Th17 and Treg cells.The findings showed that ST4 colonization did not cause any histopathological damage in normal healthy mice,and can increase the bacteria Richness and the abundance of Clostridia vadin BB60 group.In addition,ST4 colonization can activate the interleukin 5(IL-5),IL-13,and the anti-inflammatory cytokine IL-10 secreted by Th2 and Treg cells of colonic mucosa.Finally,we evaluated the effects of ST4 colonization on experimentally induced colitis.The findings showed that ST4 colonization can attenuate the inflammation of colonic tissues in mice.The colon length of the mice in the ST4-colonizd group was significantly higher than that in the control group,and the pathological score of the colon tissue was also significantly reduced compared to control mice.Further research found that Blastocystis ST4 colonization can reduce the abundance of intestinal harmful bacteria Escherichia-Shigella and Bacteroides.Flow cytometry analysis of colonic mucosal cytokines found that ST4 can increase the IL-4,IL5 and IL-13 produced by Th2 cells,and the anti-inflammatory cytokine IL-10 produced by Treg cells.In conclusion,the present study revealed the prevalence and genetic characteristics of Blastocystis in captive wild animals in Sichuan province,and also assessed the zoonotic potential of Blastocystis subtypes.Blastocystis ST4 can be asymptomatically colonized in normal healthy mice,and is beneficial to the intestinal micorbiota and the host immune system.Blastocystis ST4 colonization can help relieve the symptoms of intestinal inflammation in experimental induced colitis mice.The main mechanism is to reduce the abundance of pathobionts,such as Escherichia-Shigella and Bacteroides,and activate the host’s Th2 and Treg cells to secrete related anti-inflammatory cytokines to promote the recovery of colitis in DSS-induced colitis mice.