| Tick is a vector arthropod that can transmit pathogens to human beings.It is widely distributed worldwide and can infect and transmit various pathogens including viruses,bacteria,and rickettsia.Anaplasma is a kind of zoonotic pathogen mainly transmitted by ticks and hosted by mammals.In recent years,the continuous in-depth study of Anaplasmosis has resulted in the emergence of more and more new species of anaplasmosis and unclassified variant genotypes,which have continuously expanded the range of vector and animal host species.The whole-genome sequencing of Anaplasmosis is rapidly developing,but studies of interspecific differences and pathogenesis are scarce.Research on novel anaplasmosis has been limited to screening for infection rates,and exploration of its genome is lacking.In this study,we analyze and explore the complete genetic sequence profile and epidemiological characteristics of emerging amoebae,to understand the genetic diversity of the pathogen,host convergence,and tick-borne transmission capacity and explore the public health implications of novel tick-borne anaplasmosis.Research objectives1.To explore the existence of new Anaplasma species in Shandong province and to provide a scientific basis for the prevention of new tick-borne diseases.2.To obtain the whole genome sequence of new Anaplasma,understand its evolutionary relationship with another Anaplasmsa,and further study its genome characteristics and the function of specifically related genes.3.To understand the infection of Ca.Anaplasma cheelooensis in hosts and vectors and evaluate its public health significance based on the molecular epidemiological investigation.Research method1.Sample collection:Blood samples of ticks and animals were collected from Shandong,Guizhou,Ningxia,and other regions,including 777 ticks and 166 blood samples.Free ticks were collected from plains,hills,forests,and agricultural areas using the flag method,and parasitic ticks were captured from tick bites of fixed animals in sheep pens and slaughterhouses.According to the morphological characteristics of ticks,all tick specimens were identified at the species level and at different developmental stages.Animal blood samples are collected from the jugular vein and transported through the cold chain to laboratory storage.2.Animal host samples collected in Shandong were screened,nested PCR amplification results were used to understand the infection situation of anaplasmosis,and a phylogenetic tree was constructed to determine the evolutionary relationship of new Anaplasma.3.The whole genome draft of Anaplasma newly developed in this study was assembled by second-generation sequencing,and its evolutionary relationship in the phylogenetic tree was described according to the basic situation of the genome.To understand differences in genome size and its relation to pathogenicity by comparing genomes and the differences in homologous genes between new and related anaplasmosis species.The function of key genes of the new Anaplasma was predicted through the annotation of different databases.4.Through the design of specific primers of multiple genes of new anaplasmosis,infection rate screening of different tick samples and animal samples collected in different areas of China was conducted to understand the infection rate of new Anaplasma in tick-borne and host animals.Specific genes(16S rRNA,gltA,groEL)were amplified and phylogenetic trees were constructed to analyze the genetic evolution of different gene segments.Research results1.An unclassified Anaplasmsa was found in the blood samples of host animals after the initial screening comparison of the amplified products with the 16S rRNA universal primer.We named it Candidatus Anaplasma cheelooensis.The positive animal blood was treated with broken red cells,enriched with pathogens,nucleic acid samples were extracted,and the whole genome draft was successfully obtained by second-generation sequencing.According to the whole genome sequence,the evolutionary tree was drawn,and the Anaplasma in this study belongs to the anaplasmosis family in terms of evolutionary relationship.Anaplasmsa belongs to the same branch as Anaplasmsa phagocytophilum and Analpasma platys and is obviously different from other anaplasmosis species parasitic in red blood cells2.The whole genome characterization of the Ca.Anaplasma cheelooensis.The total size of the assembled genome was 1.13 Mb,with 92.31%genome integrity and GC content consistent with the characteristics of the anapsid family.There were 983 genes in the genome,of which 922 were putative coding sequences(CDSs)and 25 were tagged as pseudogenes.The genome size and number of genes are slightly larger than Anaplasma Capra,and the number of rRNAs and tRNAs is less than that of other Anaplasma species.Genome-wide phylogenetic analysis revealed that it is located in a separate evolutionary branch,validating it as an emerging Anaplasma species.Comparison with the genomes of six other existing Anaplasma species revealed that the anamorph family shares 590 homologous genes.The whole genome of novel anaplasma contains 174 unique homologous genes whose functions include signaling and cellular processes,metabolic processing,and genetic information processing.3.Results of the detection of infection rates in vectors and hosts for Ca.Anaplasma cheelooensis.Primers were designed based on specific fragments in whole genome sequences for screening infection rates in ticks and host animals.A total of 777 tick samples from 4 genera and 12 tick species were collected in 11 provinces and cities across China,with an overall infection rate of 0.09%.Of these,only positive samples were detected for the Haemaphysalis longicornis which was collected in Shandong provinces,suggesting that H.longicornis is its vector tick species.Testing of blood samples collected from host animals at different locations revealed Ca.Anaplasma cheelooensis infections in Shandong,Guizhou,and Ningxia,with an overall infection rate of 28.3%.Based on the systematic analysis of multiple gene fragments of newonset anaplasma in tick and host samples,a high degree of gene fragment concordance was found.Study conclusionsIn this study,a new strain of Anaplasma was identified from the blood of infected goats,its evolutionary status was clarified and a sketch of its whole genome was obtained for the first time.The total genome size was 1.13 Mb,with 983 genes.Also,by investigating its infection rate in vector and host animals,it is suggested that the Ca.Anaplasma cheelooensis may not be transmitted through different stages of the tick’s development,and the host plays a more important role after infection.Phylogenetic analyses based on three specific genes were found to be located in independent evolutionary positions.In summary,we have identified a new strain of Anaplasma in Shandong,named Candidatus Anaplasma cheelooensis,and analyzed its genomic and epidemiological characteristics.The information provided in this study will pave the way for further understanding of the invasion and pathogenesis of Anaplasma in hosts and vectors. |
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