Phylogenetic Analysis Of Anaplasma Phagocytophilum And Its Proliferation In HL60

Anaplasma spp.and Theileria spp.have become an important tick borne pathogen threatening public health in recent years.Very similar clinical symptoms of the two pathogens infection in human and animals such as fever,anemia,jaundice,thin and even death.So far,seven and six members of the genus have been reported for Anaplasma spp.and Theileria spp.,respectively.And co-infection of the two pathogens were common in clinical samples.At present,nest PCR and real-time quantitative PCR as molecular biological methods were often used to detect for the two pathogens,however,these methods detect limited certain species-specific pathogens at the species level and relatively waste the material resources and labour-intensive for detection of unknown pathogens in lots of samples.In this study,we successfully established the duplex PCR for identification the Anaplasma spp.and Theileria spp.and this method,which has a good specificity,sensitivity and repeatability,can detect the clinical samples for a initial screening at the genus level,and then identify the species-specific pathogens at the species level.This method we established can save time,effort and cost and provide the technical support for the epidemiology investigation and pathogen identification of Anaplasmosis and Theileriosis.329 blood samples of dogs,sheep and cattle were collected and detected for Anaplasma spp.and Theileria spp.Then the phylogenetic analysis of AP,a kind of a kind of zoonosis pathogen with great significance of human public health security,was performed.That provide a base of molecular epidemiology and population genetics of it.AP,which was transmitted by vector ticks,as obligate intracellular organisms,was usually reproduced in the host cell.Different cell lines for the isolation and culture of AP have broke through the limitations of indirect study and molecular biology method research of it.Although the AP as a kind of important mode pathogens,however,the infected host and intracellular survival mechanism need further study,and the proliferation regularity of AP in cell has not been reported.In this study,we successfully established the fluorescence quantitative PCR based on 16 s r RNA genes of AP,and this method with high specificity,sensitivity and repeatability did provided a technical support for identification on pathogens,quantitative detection and in the early rapid diagnosis of the AP.We successfully cultivate AP derived from sheep in HL60 cell,monitor the dynamic changes of the AP infection in HL60 by fluorescence quantitative PCR established in this study,preliminarily clearing the quantitative change rule of AP in HL60 and providing the basics of the reproduction regulation and pathogenic study of AP.1 The establishment of duplex PCR of Anaplasma spp.and Theileria spp.and Phylogenetic analysis of AP.In order to preliminary screen the unknown pathogens in lots of samples,we have established the duplex PCR for identification the Anaplasma spp.and Theileria spp.at genus level for providing a technical support for identification on pathogens,epidemiological study,the prevention and control of disease.We have designed genus species premiers AR1/AF1 and Tr/Tf based on the conserved DNA regions of 16 S r RNA and 18 S r RNA of Anaplasma spp.and Theileria spp.respectively by Primer5.0 software.After optimizing condition,specificity,sensitivity and repeatability test,the results show that this duplex PCR has a good specificity,sensitivity and repeatability.Several primers from the published were used for comparison of the efficiency of the duplex PCR detection capability,and a total of 50 blood samples were collected randomly from a sheep farm in the Guizhou Province of China in 2015.The results show that the duplex PCR can be used for for clinical detection of the Anaplasma spp.and Theileria spp.In order to further understand the prevalence of AP,we preliminary screen the clinical samples by duplex PCR established in this study.243 blood samples from dogs,51 blood samples from sheep,35 blood samples from cattle were collected for identification the infection of AP,following identification the species-specific pathogens by species premiers.The results indicated that the prevalence of Anaplasma spp.from dog blood samples was 13.6(33/243),and none of these samples was positive for Theileria spp.The positive rate of co-infection of Anaplasma spp.and Theileria spp.in sheep blood samples was 88.2%(45/51).The infection rate of Anaplasma spp.in sheep blood samples was 92.2%(47/51),and AP of them was 80.4%(41/51).The prevalence of Theileria spp.of sheep blood samples was 88.2%(45/51).25.7%(9/35)and 22.9%(8/35)were detected for Anaplasma spp.and Theileria spp.in cattle blood samples,respectively.The results showed that the co-infection Anaplasma spp.and Theileria spp.were common in blood samples.In order to understand the phylogenetic analysis of AP among different host,further clearing genetic diversity of AP,that provide a base of molecular epidemiology and population genetics of AP.We did the sequence alignment and phylogenetic analysis of AP isolated from dogs based on the AP 16 S r RNA gene for providing basis of the research on potential transmit risk among dog-human-livestock.In this study,dog isolates in this region(KX236049-KX236052)were in a same clade with sheep isolates(KU321305 and KU321306),indicating the close relationship among them.The dog isolate(KX236049)shared 99% and 100% similarity with an American human isolate(NR044726)and an sheep isolate(KX321305).The results showed that the dog isolate of AP potential zoonotic risk.The blood samples from cattle herd with disease were infected through microscopy observation of cattle blood smears stained with Giemsa staining and PCR,further examination on identification tick species and carrying pathogens.The results showed that A.marginale and T.annulata were observed under microscope and the two pathogens DNA were identified.Ticks feeding on cattle were collected and identified as R.microplus,In addition,the A.marginale msp4 sequence obtained from R.microplus(KX904527)shared 100% similarity with that of cattle strain(KX840009)and they were classified into a cluster in this study.The results indicated that A.marginale found in cattle may be transmitted by the R.microplus feeding on them.2 Development real time fluorescent quantitative PCR for detection of AP and its proliferation in HL60In order to establish a rapid,sensitive,specificity and quantifiability detection method of AP,a pair of primers of fluorescent quantization PCR was designed according to the conserved regions of AP 16 S r RNA gene.A expected size fragments was amplified by nested PCR with the two sets primer of EE1/EE2 and 1-25F/183-205 R.Construction of a recombinant plasmid containing 16 S r RNA gene was as template to set up RT-PCR standard curve.The method of RT-PCR for detection AP was established,meanwhile specific assay,sensitivity assay,reproducibility of the assay clinical application were determined.The results indicated that standard curve showed a good linear relationship with template concentration ranging from 6.4�103~ 6.4�108copies/?L.Then,the specific and sensitive tests showed that the assay was only detected A.phagocytophilum with the limit detection of 6.4 �102 copies/?L,but had no cross-reaction with A.ovis,A.bovis,T.annulata and sterilization dd H2 O.The variations of both inter-and intra-assay were less than 2%.The detection rate of this method is high than convention PCR assay(16.67%)for detecting 30 clinical samples.The method was applied for the qualitative detection of AP,and it was developed the basis for and quantifiability detection and rapid diagnosis of infection in the early of AP.In this study,AP derived from sheep was cultivated in HL60 cells and verified its infenction by microscopy observation blood smears stained with Giemsa staining,n PCR,quantifiability PCR and IFA.The results showed that fuchsia pathogens in HL60 were observed under microscope in DPI 5,and the HL60 were infected of AP by n PCR and sequencing.In IFA test,the immunofluorescence were observed in the cytoplasmic of HL60 infected of AP,none of in cytoplasmic HL60 uninfected AP.In addition,the dynamic change of AP in HL60 was monitored by quantifiability PCR.The results showed that the copies of AP in HL60 fell sharply in DPI5,following rising sharply,and the peak of copies was appeared in DPI13,following felling.The copies of AP reaches a stable state until DPI28.This study preliminary clear the quantifiability changing regulation of AP in HL60,providing the basics of the reproduction regulation and pathogenic study of AP.The sequence alignment and phylogenetic analysis based on 16 S r RNA gene of AP derived from sheep and cultivated in HL60 cell.The results showed that this AP isolate from HL60(MH119059)shared 99.3% similarity with four Europe and America AP dog isolates(JX173651,U10873,CP006618 and JX173652),two human isolates(AF093788 and NR-044762),and all of them fell into the same clade in the phylogenetic tree.Also,the isolate(MH119059)were grouped in the same clade as custer 1 with a sheep isolate(KU321301),two rat isolates(GQ413337and KC470064)from China.The results showed that the isolate(MH119059)was potential zoonotic risk.