Investigation Upon The Isolation And Genomics Of Ehrlichia Canis,and Establishment Of Canine Model Infected E.Canis

Ehrlichia,belonging to family Anaplasmataceae,is the obligate intracellular Gram-negative bacteria with wild spectrum of infecting host.Ehrlichia organisms are mainly transmitted through the bite of an infected tick.There are five species in the Ehrlichia genus based on the sequences of 16S rRNA gene and other related genes,mainly E.canis,E.chaffeensis,E.ruminantium,E.ewingii,E.muris.Human monocytic ehrlichiosis(HME)is caused by the E.chaffeensis which is the most important agents together with E.ewingii for human ehrlichiosis.The heartwater in ruminants,including cattle,sheep,goat,caused by E.rumiantium,is one of the most important organism in farm animals that will decreas the economic ability of the animals.E.canis is an zoonotic pathogen mainly caused canine monocytic ehrlichiosis(CME)in canine animals with acute,subclinical and chronic phase.1.Establishment of Ehrlichia special FRET-qPCRThe Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens.There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction:E.canis,E.chaffeensis,E.ewingii,E.muris and E.ruminantium.We developed primers and probes against the 16S rRNA gene to enable us reliably detect the five major Ehrlichia spp.in a single FRET-qPCR.We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from the blood of domestic ruminants from five Caribbean islands.The Ehrlichia present were determined using melting point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the citrate synthase gene(gltA).Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A.phagocytophilum but gave positive reactions with reference strains of the most generally recognized species and with other less characterized Ehrlichia of domestic ruminants,mainly E.ovina,the Panola Mountain Ehrlichia,and Ehrlichia sp.BOV2010.Melting point analysis revealed 4 distinct groups:E.ruminantium(Tm?55.8?);E.chaffeensis and E.ewingii(Tm?57.7?);E.canis,E.uris,E.ovina and Ehrlichia sp.BOV 2010(Tm?62.0?);and the Panola Mountain Ehrlichia(Tm?65.5?).The detection limit of the FRET-qPCR was single copy in a reaction and the sequences of the FRET-qPCR products were as expected.With DNA from domestic ruminants from the Caribbean we found 12.2%(134/1,101)positive:cattle(76/385;19.7%),sheep(45/340;13.2%)and goats(13/376;3.5%).Melting point analysis and sequencing of the FRET-qPCR and nested PCR of gltA products showed the Ehrlichia we detected were E.canis or very closely related organisms.In a single reaction,our Ehrlichia FRET-qPCR can detect the Ehrlichia spp.we studied and differentiate them into four groups.Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia,possibly E.canis or very closely related organisms.2.Molecular epidemiology of ehrlichiosis in dogs and ruminants from ChinaRecords of pet and guard dogs and ruminants in China can be found dating 8,000 and 10,000 years ago,respectively.With improvement of quality and quantity of food animals and companion animals in China,the zoonotic pathogens carried by them also put the master into a more danger situation.The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important pathogens for human and animal.There are five generally recognized species,including E.canis,E.chaffeensis,E.ewingii,E.muris and E.ruminantium.As one of the vector-borne agents that are potential zoonoses and cause substantial morbidity and mortality worldwide,there are limited data on these organisms in China.Quantitative PCRs for Ehrlichia spp.were performed to investigate their prevalence in convenience whole blood samples obtained from 1,114 dogs from 21 veterinary clinics and a commercial dog breeding facility in ten provinces of China.In addition,the PCRs were performed on 146 Rhipicephalus sanguineus senso lato and 37 Linognathus setosus collected from dogs in the commercial dog breeding facility.16S rRNA FRET-qPCRs were used to screen convenience whole blood samples from 2,240 domestic ruminants in 12 provinces of China for Ehrlichia spp.Positive samples were further analyzed with a standard PCR for the gltA gene.DNAs of Ehrlichia canis(1.3%,15/114)were detected in the bloods of the dogs studied,and in R.sanguineus senso lato(4.1%,6/146).Ehrlichia spp.DNA was detected in sheep(1.8%,2/111),goats(1.1%,3/270),and cattle(3.6%,65/1,830)but not in water buffaloes(0/29).3,In vitro isolation and culture of Ehrlichia canisCanine monocytic ehrlichiosis caused by the bacterium Ehrlichia canis with the tropism for canine monocytes and macrophages is an infectious,non-contagious,tick-borne disease of domestic dogs and some wild canidae.The diasease was first described in Algeria and has a worldwide geographical distribution which coincides with the distribution of the vector ticks.Ehrlichiosis in dogs was first described in South African in 1938 and was first reported in China 61 years later.E.canis was first propagated in monocyte cell cultures derived from the peripheral blood of acutely infected dogs further improved this method by adding E.canis-positve monocytes to uninfected monocyte cultures obtained from healthy donor dogs.This changed when cells of a dog suffering from malignant histiocytosis gave rise to a continuous cell line,namely DH82 which was then successfully used to continuously propagate E.canis in vitro at 37?.Other heterologous host cells were used for in vitro cultivation of E.canis,including human-canine hybrid cells,immortal human microvascular cells,and a continuous BALB/C mouse macrophage cell line.The successful in vitro isolation and propagation of E.canis also performed in Ixodes scapularis-derived IDE8 tick cell,Ixodes sccapularis cell line ISE6,and Ixodes ricinus-derived cell line IRE/CTVM18.Here,we isolated and passaged the E.canis from leucocytes of the positive dog with DH82 cells and Vero cells.With the Giemsa stain,we visualized the number and morphological difference of introcellular membrane-bound vacuoles containing morulae in DH82 cells and Vero cells post-infected E.canis 1,2,and 3 days.4.Whole genomic sequencing and comparative genomics of Ehrlichia canis str.YZ1Ehrlichia canis,a small obligately intracellular,tick-transmitted,gram-negative,is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis.Complete genome sequecing revealed that the E.canis strain YZ1 consists of a single circular chromosome of 1,314,789 bp predicated to encode 1,022 genes,36 transfer RNA with transferability of 20 amino acids,single 5S rRNA,16S rRNA and 23S rRNA.There are 65 interspersed nuclear elements and 158 tandem repeats,and genomic island sized 44.886 bp in the E.canis YZ1 genome with%GC of 29.0%.After functional analysis on the genes of E.canis Strain YZ1,there are 2,959 GO annotations mainly focus on cellular process and metabolic process,455 proteins encoded by the genes related to the KEGG metabolic pathways with 74 focus on the translation in genetic information processing,757 amino acid sequences related to the COG database with 135 focus on the traslation/ribosomal structure and biogenesis.We done the colinear analysis of E.canis YZ1 with other six reference genome sequences of Ehrlichia,including E.canis strain Jake,E.ruminantium,E.chaffeensis,E.muris,E.mineirensis and Ehrlichia sp.We found that only the referee genome of E.canis strain Jake give the high similarity to ours but not other five ones.Furthermore,the E.canis Jake used in this study as the reference sequence has the least SNP locus which is the DNA polymorphism produced by the mutation of single nucleotide.And there are !ots of insertion and deletion of nuclotides in the CDS of genome of E.canis strain YZ1 studied in this study.Meanwhile,the inversion and translocation mutation of genes were found in the very beginning and terminal of the genome of E.canis strain YZ1 used in this study.5.Preliminary study on the founction of autophagy in cells infected Ehrlichia canisEhrlichia canis is an oligatory intracellular bacterium that causes a potentially fatal emerging zoonosis,canine monocytic ehrlichiosis(CME).Adaptation to a life in eukaryotic cells and transmission between hosts has been assisted by the deletion of many genes that are present in the genomes of free-living bacteria(including genes required for the biosynthesis and metabolism).However,the mechnism of the entrance pathway of E.canis and relationship between autophagy of host cell and E.canis infection is still unknow.We found in this study that the concentration of autophagy maker protein,LC3 and p62,has increased and decreased much in DH82 cell(canine macrophagocyte)and Vero cell(simian kidney cell)infected with E.canis,respectively.Under the fluorescence microscope,we found punctiform fluorescence of the LC3 and RAB5 in the membrane containing the E.canis after transfecting with LC3-GFP plasmid and incubating with primary antibody for RAB5 and secondary antibody.The bacteria of E.canis per cell increased after treating with rapamycin(target on the mTOR protein),bafilomycin(target on the lysosome)and 3-MA(target on the PI3K protein).E.canis inclusions retain characteristics of the early endosome,including the markers RAB5,and fuse with lysosomal receptors.E.canis can use the autophagy for the pathogen replication and multiplication with the nutrients from the host cell cytoplasm.6.Establishment of canine model infected with Ehrlichia canisEhrlichia canis is a Gram-negative,highly pleomorphic and obligate intracellular bactrium,which is enveloped with a rippled thin outer membrane.Canie monocytotropic ehrlichiosis(CME),caused by E.canis,is an important canine disease with a worldwide distribution.The disease was initially identified in Algeria in 1935.Later in 1999s the agent was first detected in military dogs from China.In order to comprehend deeply about the E.canis infection,we established the canine model with Beagle dog by supervising the clinical signs and characteristic hematological and biochamical abnormalities.The acute phase presented right after the first infection of E.canis was characterized with high fever,depression,lethargy,anorexia,severe nasal and ocular discharge.During the acute stage,severe thrombocytopenia was found,and leukocyte,lymphocyte,monocyte and granulocyte counts were mildly reduced.Mild increases in alkaline phosphatase(ALP)were frequent in the serum of dogs infected with E.canis during the acute phase.The cytokines of serum from differnet stage of infection dogs was no significanrly difference.The concentration of canine cytokines of CD8B,IL17A and IL10 increased heavily in lymph nodes,spleen and liver from the infected dogs.The copies of E.canis nucleotides in the blood of infected dogs curved after 17-day after inoculation with the pathogens,and keeped in the body with similar level.Both the doxycycline and rifampicin antibiotics used in this study were positive to remit the clinical characteristics,but the treatment of doxycycline was active and could remove the agents from the pathogen-infected dogs.Histophathology of the liver revealed mild degeneration in hepatocytes,lymphocytic cellular infiltration,sinusoidal dilatation and hyperaemia.Prominent mononuclear cellular infiltration in the interalveolar septa and vasculitis were observed in the lung.Lymphocytic cellular infiltration was also observed in the spleen and kidney of the dogs infected organism of E.canis.The spleen is a highly perfused organ involved in the immunological control and elimination of E.canis pahtogens.