| BackgroundStreptococcus intermedius belongs to the S.anginosus group(SAG)that includes S.constellatus and S.anginosus.It is part of the normal oral cavity and upper respiratory tract flora,as well as those of the gastrointestinal and female urogenital tracts.However,S.intermedius may cause human infections,usually monomicrobial,notably purulent abscesses involving the liver,lungs,psoas,spine and central nervous system,and infective endocarditis.This bacterium was first described by Guthof in 1956 after being isolated from dental abscesses.And then the role of S.intermedius in human infections diseases is increasingly being reported.Patients with invasive S.inter,medius infections have significantly longer hospital stays and higher mortality than patients with other S.anginosus group infections,suggesting that identifying this species might be important for the management of patients.The genus Anaplasma belongs to the family Anaplasmataceae,in the order Rickettsiales,and comprises different species:Anaplasma marginale,Anaplasma centrale,Anaplasma ovis,Anaplasma phagocytophilum,Anaplasma bovis and Anaplasma platys,Anaplasma capra and Anaplasma caudatum.Anaplasma spp.are causative agents of tick-borne diseases(anaplasmosis)with a remarkable impact on human and animal health.The effects of anaplasmosis on the health and productivity of domestic animals have been known for over a century,and anaplasmosis is still today an important cause of economic losses in livestock farming.Anaplasma phagocytophilum,A.capra and A.ovis can cause serious diseases in humans.Vertebrate hosts are considered reservoirs of these bacteria because they can develop persistent infections and act as a source of infections for the tick vectors.When ticks feed on infected hosts,Anaplasma spp.enter into the tick where they can replication.Then the bacteria migrate to salivary glands of tick,in which they enter the saliva when the tick feeds on the next animal host.A large number of publications have reported the detection of Anaplasma spp.in different species of ticks of different regions.Currently,recognition of Anaplasma as a genus of public health significance is more recent and has contributed to rising interest about these bacteria,resulting in more information about their genetics and pathobiology.Hantavirus(HV)mainly causes two diseases:Hemorrhagic fever with renal syndrome(HFRS)and Hantavirus pulmonary syndrome(HPS).The main hosts of HV are rodents and insectivores.It can cause persistent recessive infections of the host through a lifetime.The human may infect the virus through inhalation of the excrement of host animal carrying the virus,such as aerosol of urine,feces,saliva,or eating the infected food,or getting bite by the host animal.HV is host-specific,it has co-evolved with its host animals,such as the mouse subfamily-related Hantaan virus(HTNV),Seoul virus(SEOV),and Dobrava virus(DOBV),which can cause HFRS,mainly distributed in Asia and Europe;the voles subfamily-related Puumala virus(PUUV)and Prospect Hill virus(PHV),mainly distributed in Europe and Asia,which can also cause HFRS;The cotton-white subfamily-related Sin Nombre virus(SNV)and Andes virus(ANDV),mainly distributed in the Americas,which can cause HPS;the insectivore-related Thottapalayam virus(TPMV)is globally distributed with little known about it's pathogenicity.HFRS continues to occur worldwide,thus become a serious worldwide public health problem.Currently,it is estimated that 150,000-200,000 HFRS cases occur each year worldwide,of which 70-90%occurred in China.Hantavirus cases are increasingly reported in the United States and Europe.ObjectiveTo investigate the prevalence of tick-bone Anaplasma in ticks from Shandong Province.To investigate the prevalence of HV in shrews and rodents,and to determine whether MJNV can cause infectious disease to human.To describe the genetic diversity and pathogenesis substratum of S.intermedius.Methods1.Pan-genome analysis of S.intermedius(1)Streptocococcus intermedius strains were isolate from patients with different infectious disease.The other genome sequences were download from GenBank database.(2)The obtained S.intermedius strains were identified by MALDI-TOF MS and then the genomic DNA(gDNA)of S.intermedius strains was extracted.(3)gDNAs were sequenced using a MiSeq sequencer(Illumina,Inc,San Diego CA 92121,USA)with the Paired-End and barcoding strategy.The obtained sequencing reads were assembled by A5 assembler with default parameters and then online tool BLAST from NCBI to search the contigs with database to remove the contaminations.Whole genome was annotated using Prokka software with default parameters.The AGIOS(Average of genomic identity of orthologous gene sequences),OrthoANI(Average nucleotide identity,ANI),and online tool GGDC(Genome-to-Genome Distance Calculator)were used to analyze the similarity of genomic sequences.VFDB was used to identify the virulence factors of S.intermedius.The online tool ResFinder and ARG-ANNOT database were used to identify the resistance genes.The online tool CRISPRCasFinder was used to identify the CRISPRs.The pan-genome analysis was performed using the softwares GET HOMOLOGUES,Roary,and Proteinortho,respectively with the parameters of identity ?70%,coverage>70%.The Clusters of Orthologous Groups(COG)database was used to find the function of genes.The Blast atlas and creating a circular comparison was done using online GView Server with S.intermedius strain ATCC 27335 as the reference genome.2.Collection and detection of animal specimens(1)Ticks were collected by flagging over vegetation using a 1 m2 flannel flag in rural areas of Huangdao District of Qingdao City in Shandong Province,from June to July 2013.To extract DNA the ticks were pooled,with each pool containing 40 larval ticks,20 nymphal ticks,or 5 adult ticks.The DNA extracted from the ticks was amplified by PCR for Anaplasma species genes.The sequences were analyzed with Blast in the NCBI database.Based on the PCR positive results,the minimum positive infection rate of ticks was calculated.(2)The glt A,groEL,msp4 and msp2 genes of A.capra were also identified by PCR.We performed a phylogenetic analysis based on gltA,groEL,msp4 and msp2 genes sequences of A.capra to identify their phylogenetic affiliations with other isolates of the genus Anaplasma using MEGA5.0.For constructing the phylogenetic tree,the following options were used:Maximum Likelihood Method,"Kimura 2-parameter model" model,"use all sites" parameter,"1 St+2+3+Noncoding Sites",1000 replicates of the bootstrap test.(3)Rodents and shrews were trapped once in the middle of each month(except for September)for 3 consecutive days using snap traps with peanut bait in Huangdao District in 2014.The blood,liver,spleen,lung,kidney tissues of the rodents and shrews were collected aseptically and frozen at-80? until use.We recorded the basic information about the shrews and rodents such as sex and appearance.Shrews and rodents were classified according to appearance(hair color)and body structures.All shrews were further confirmed by molecular typing described below.For amplification of MJNV in shrews and rodents,outside and inside primers for RT-PCR were designed from the S and L segments of the MJNV genome in this study.We used one step RT-PCR and nested-PCR.3.A preliminary analysis of the pathogenicity of MJNV.We collected acute phase serum from patients with suspected HFRS and the total RNA in the patient's serum was extracted.RT-PCR was used to detect MJNV in the patient's serum.The amino acid sequences of SEOV and MJNV nucleocapsid proteins were compared to find the specific amino acid parts of the two viruses,and a prokaryotic expression vector was constructed to express and purify partial fragments of nucleocapsid protein.Results1.Thirteen strains G1552-61557 and G1562-61568 were isolated from patients with different symptoms of infection from August 2014 to November 2016.Fourteen S.intermedius genomes were acquired from GenBank.The dDDH values ranged from 80.5 to 99.3%between all 27 strains which confirm that strains belong to S.intermedius species.The OrthoANI values ranged from 97.78%to 100.00%which corresponds to the dDDH values.AGIOS results(range between 50.96%to 77.10%)confirmed the high level of sequence identity among all 27 strains.In all 27 S,intermedius strains,nearly 252 virulence factors were identified in total.Sixty-nine core virulence factors of them were shared by all strains and 79 unique virulence factors present only in one strain.We did not find significant association between virulence genes and diseases.CRISPRs analysis in S.intermedius strains showed that 14 of 27 strains contained CRISPRs.Three of the 14 strains(G1564,G1565,631 SCON)had more than one CRISPRs for a total of 17 CRISPRs identified within S.intermedius.A set of prophage elements was identified in all 27 strains.Tetracycline resistant gene(tet(M))were identified in strains G1552,C270,KCOM1545,G1555,LC4,30309 and 3281 1;Tetracycline resistant gene(tet(32))was identified in strain 631_SCON;Macrolide resistant gene(erm(B))were identified in strains G1552,C270,G1555 and 30309.2.A total of 1355 core genes were determined in S.intermedius.The average proportion of core genes corresponds to 72%per strain.A total of 1054 strain-specific genes were identified in S.intermedius and the average number of strain-specific genes is 39.A total of 1611 accessory genes that were shared by two or more strains were identified in S.intermedius.Our calculations suggest that the S.intermedius pangenome size is 4020.The distribution of proteins in COG categories was quite similar in all 27 strains.Approximately 79.72%of all proteins coded in all strains were identified in COGs superfamily.The most abundant sub-categories were related to carbohydrate transport and metabolism(G)and translation,ribosomal structure and biogenesis(J).3.Species composition and infection status of captured ticksA total of 3,300 Haemaphysalis longicornis ticks were collected from June to July 2013 in Huangdao District,Qingdao City,Shandong Province,including 1,620(49.09%)adult ticks,1,560(47.27%)nymphal ticks,and 120(3.64%)larval ticks.The sequences of 16S rRNA,groEL,glt A,msp2,and msp4 genes were obtained by PCR amplification from one nymphal tick pool(minimum infection rate 0·06%,1/1560)and seven adult tick pools(minimum infection rate 0·43%,7/1620).DNA sequence homology of the genes between the Anaplasma species in ticks from Shandong Province and "A.capra" was 99-100%.The 16S rRNA sequences of A.bovis were also obtained from 69 tick pools(minimum infection rate 2.09%,69/3,300),including 46 adult tick pools(minimum infection rate 2.84%,46/1,620),22 nymphal tick pools(minimum infection rate 1.41%,22/1560)and one larval tick pool(minimum infection rate 0.83%,1/120)4.Species composition and infection status of capture shrews and roednts(1)From January to December 2014,a total of 178 shrews(54 males,30.34%and 124 females,69.66%)were captured in Huangdao District.Among the shrews,164 were Crocidura lasiura(92.13%),7 were Crocidura attenuate(3.93%),and 7 were Crocidura shantugensis(3.93%).A total of 475 rodents(176 males,37.05%and 299 females,62.95%)were tested,which was captured from January to December 2014(except for September).The rodents included seven species with 163 striped field mice(Apodemus agrarius,34.32%),102 house mice(Mus musculus,21.47%),88 brown rats(Rattus norvegicus,15.3%),62 greater long-tailed hamsters(Cricetulus triton,13.03%),29 Chinese hamsters(Cricetulus griseus,6.11%),29 Chinese striped hamsters(Cricetulus barabensis,6.11%),and 2 buff-breasted rats(Rattus flavipectus,0.42%).(2)We first screened insectivore-borne MJNV and rodent-borne HTNV and SEOV in the captured shrews.We found that 19 of 178(10.6%)shrews,including 18 C.lasiura shrews and 1 C.shantugensis shrew were positive to MJNV.We also amplified RNA sequences from 2 of 178(1.1%)shrews(C.lasiura)with HTNV and SEOV common primers,and DNA sequencing showed that both sequences wereSEOV.No shrew was co-infected with both MJNV and SEOV.The prevalence of MJNV infection among shrews had no statistical difference between different sexes(p=0.514).We then tested for insectivore-borne MJNV on captured rodents and found that none of the 475 rodents was positive to MJNV.5.Successfully construct prokaryotic expression vector,express and purify partial fragments of MJNV and SEOV NP.Conclusion1.The pan-genome size of 27 S.intermedius strains was 4020,including 1355 core genes,1611 accessary genes,and 1054 strain-specific genes.The pan-genome model of S.intermedius is open.2.The distribution of proteins in COG categories was quite similar in all 27 strains,but there are differences between core genes and strain-specific genes.No statistical association was found between genome size,G+C content,prophage sequences,CRISPRs,resistance genes,and infection types.3.The dominant species of ticks in the wild areas of Huangdao District in Shandong Province is Haemaphysalis longicornis.The tick carries A.capra,a new pathogenic Anaplasma species.4.The minimum infection rate of A.capra.in Haemaphysalis longicornis was 0.24%.5.We demonstrated that MJNV is highly prevalent in shrews collected in Shandong Province,and we detected SEOV in the shews,but not HTNV in shrews,suggesting that the major animal hosts of Imjin virus are shrews;and SEOV can infect both rodents and shrews. |
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