| Anaplasma phagocytophilum belongs to the genus Anaplasma,which is an obligate intracellular and zoonotic pathogen transmitted by ticks.This pathogen is widely distributed all over the world and has a wide host spectrum.It is considered to be an emerging pathogen with important public health significance,which cause clinical symptoms such as fever,inappetence,dullness and a fall in milk production.With the progress of research on the genetic diversity of A.phagocytophilum,different strains were identified based on the 16 S r RNA gene.However,the relationship,pathogenesis and the prevalence and distribution of different A.phagocytophilum isolates in animals from China are still unclear.Therefore,the prevalence of different A.phagocytophilum isolates from goats,sheep,cattle,dogs and Ixodes ticks in 9 provinces in China was investigated in this study.And on this basis,the main epidemic A.phagocytophilum-like1 was cultured in vitro and the analysis of differentially expressed proteins of HL-60 cells induced by A.phagocytophilum-like1 in order to assess the public health risk of A.phagocytophilum-like1 and the interaction between A.phagocytophilum and its host,and provide a theoretical basis for studying new targets of antimicrobial therapy or blocking pathogenic pathway and the prevention and control of zoonotic Anaplasmosis in this study.To identify the prevalence of A.phagocytophilum and genetically related strains in different animals in China,blood DNA samples of 1031 goats and 307 sheep,286 cattle,261 dogs and 277 tick pools DNA samples consisting of 751 H.longicornis collected in goat from some provinces were investigated and phylogenetic analysis was performed based on 16 S r RNA combined with RFLP.To verify the genotyping results based on 16 S r RNA gene of A.phagocytophilum,positive samples were tested based on gro EL gene and evolutionary analysis was performed The results showed that A.phagocytophilum and genetically related strains were detected in 313(23.39%)of 1338 goats and sheep blood samples.The prevalence was higher in goats(27.64%,285/1031)than in sheep(3.97%,11/277).And the positive rate of H.longicornis was 3.97%(11/277).The total positive rate of A.phagocytophilum and genetically related strains in cattle blood DNA samples was 11.53%(33/286);and in dog blood DNA samples,8positive samples(3.01%)were detected.The different A.phagocytophilum isolates were identified by Xcm I and Bsa I restriction enzymes.The results showed that A.phagocytophilum-like1 and A.phagocytophilum-like2 were found,and the positive rates were 22.65%(303/1338)and 0.75%(10/1338)in goats and sheep blood samples,respectively,of which A.phagocytophilum-like2 was only detected in goats,and no mixed infection was found in blood samples of goats and sheep.In addition,only A.phagocytophilum-like1 was detected in H.longicornis.Anaplasma phagocytophilum,A.phagocytophilum-like1 and A.phagocytophilum-like2,were found in cattle blood samples,with positive rates of 2.80%(8/286),11.53%(33/286)and 3.14%(9/286),respectively.The mixed infection of the two isolates was observed.The positive rates of mixed infection were 2.80%(8/286)for A.phagocytophilum and A.phagocytophilum-like1,and 3.14%(9 /286)for A.phagocytophilum-like1 and A.phagocytophilum-like2.The 8 dog positive samples were all A.phagocytophilum-like1.The phylogenetic analysis results were consistent with the enzyme digestion results.The obtained sequences were located in the A.phagocytophilum-like1 and A.phagocytophilum-like2 clades,respectively,in evolutionary tree based on the gro EL gene.However,the gro EL gene sequence of A.phagocytophilum was not amplified in this study.Our findings provide additional theoretical data on the molecular epidemiology and genetic diversity of A.phagocytophilum.In order to observe the combination,invasion and the development process of A.phagocytophilum-like1 in HL-60 cells,the A.phagocytophilum-like1 positive goat blood was collected to separate neutrophils.The separated neutrophils were inoculated into HL-60 cells for culturing pathogen.The infection and morphology of A.phagocytophilum-like1 in HL-60 cells were observed by PCR method targeting the 16 S r RNA gene,Giemsa staining,indirect immunofluorescence,and transmission electron microscopy.The proliferation of A.phagocytophilum-like1 within HL-60 cells was monitored by q PCR,in order to determine the optimal time point for purification of pathogen from infected HL-60 cells.Transmission electron microscopy was used to monitor A.phagocytophilum-like1 binding,invasion,and intracellular development in HL-60 cells.The results showed that the DNA samples of HL-60 cells detected by PCR were positive for A.phagocytophilum-like1,indicating that HL-60 cells were successfully infected.The morula of A.phagocytophilum-like1 was observed within HL-60 cells by Giemsa staining,indirect immunofluorescence and transmission electron microscopy.Thirteen days after infection was the optimal time for separation and purification of pathogen using q PCR to monitor the proliferation of A.phagocytophilum-like1 within HL-60 cells.Transmission electron microscopy showed that dense-cored cell(DC)adhered the cell surface by 2 hours post infection.By 4 hpi and 8 hpi,the internalization of DCs into the cell membrane was observed and a part of internalized DCs detached from the cell membrane.By 12 hpi,it was observed that the A.phagocytophilum-like1 vacuole became larger and some DCs began to become larger and transformed into reticulate cell(RC).By 24 hpi,RCs was found to have replicated.By 36 hpi,48 hpi and 72 hpi,two forms of DCs and RCs were present within the cells,indicating that re-infection of the pathogen occurred after 36 hours postinfection.This study laid a foundation for the pathogenic research and effective prevention and control of A.phagocytophilum-like1.In this study,we compared the proteomes of HL-60 cells infected A.phagocytophilum-like1 for 12 and 48 hours with uninfected HL-60 cells using 4D LQ quantitative proteomic.Taking 1.5-fold as the threshold for differential expression changes,a total of 86 differentially expressed proteins were identified,among which 22 were up-regulated and 64 were down-regulated by 12 hours post infection.There were 123 differentially expressed proteins,of which 64 were up-regulated proteins and 59 were down-regulated proteins by 48 hpi.Comparing 48 hpi and 12 hpi,there were 178 differentially expressed proteins in host cells,of which 65 were up-regulated and 113 were down-regulated.The differential protein expression results were verified by PRM.Through functional analysis of differentially expressed proteins,it was found that up-regulated proteins of HL-60 at 12 hpi were mainly involved in apoptosis,receptor-mediated endocytosis,autophagy,protein and lipid transport,and down-regulated proteins were mainly involved in cellular Skeletal components,cell adhesion,and cell surface pathways.By 48 hpi,up-regulated proteins of HL-60 cells were mainly involved in fatty acid biosynthesis and metabolism,lipid biosynthesis and metabolism,amino acid biosynthesis and other processes,while down-regulated proteins are mainly involved in apoptosis,immune response and other processes.Comparing the differential proteins at 48 hpi and 12 hpi,the proteins related to cytoskeleton,cell adhesion,chemotaxis and defense response were up-regulated;the proteins related to apoptosis were down-regulated.This research was helpful for the study of pathogen-host interaction and invasion mechanism. |
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