| BackgroundTicks are important vector for the transmission of zoonotic diseases,because they sting the host and suck blood,which not only causes blood loss,ulcers form at the stab wound,but also causes allergic reactions and toxin damage.More importantly,it can transmit viruses and bacteria,spirochetes,rickettsia and protozoa and other categories of pathogens,with unique medical significance.In recent years,the geographical distribution of vector ticks has expanded,and new tick-borne diseases have continued to occur,which has become a new threat to global public health.Northern China has always been an important epidemic area for tick-borne diseases such as forest encephalitis,Lyme disease,Xinjiang hemorrhagic fever and spotted fever.Tick-borne protozoosis is a chronic wasting disease.The pathogen mainly parasitizes in red blood cells.Humans and animals infected with protozoa will have anemia,jaundice,fever,headache or myalgia,and other clinical symptoms,which are life-threatening in severe cases.The common tick-borne protozoa include Babesia spp.,Theileria spp.,Colpodella spp.,etc.They are widely distributed and many of them are important zoonotic pathogens.However,the research on other tick-borne protozoa is relatively scarce,except for Theileria,which is widely studied in livestock.ObjectiveTo understand the carrying and infection of neoprotozoa,especially Babesia and Colpodella,among people,host animals and vectors of tick-borne diseases in northern China,and to grasp the risk factors,so as to provide scientific basis for the prevention,treatment and control of new human blood protozoa.Methods1.Population survey and sample collection:first,the spot monitoring survey of patients bitten by ticks in sentinel hospitals.Relying on the sentinel Hospital of the forestry center in Mudanjiang City,Heilongjiang Province,the spot will collect blood samples from patients who have a clear history of biting,fill in the epidemiological questionnaire,and carry out the epidemiological survey of people infected with tick borne protozoa in northern China;Second,retrospective screening of retained blood samples in the laboratory department of sentinel hospital in the epidemic area;The third is the follow-up investigation of suspected patients in the sentinel Hospital of the fifth medical center of the PLA General Hospital,namely,follow-up investigation of suspected patients with clinical symptoms including hemolytic anemia,excluding other related diseases.2.Collection of samples of host animals and vector ticks:after collecting blood samples from people,the investigation of host animals and vector ticks in tick borne disease distribution epidemic areas shall be carried out at the same time.The blood samples of free ticks,parasitic ticks of host animals and host animals in the living environment were collected around patients in northern China for detection.3.Molecular biology detection:the target fragment is amplified by Nest PCR and quantitative PCR amplification technology,and the collected blood and tissue samples are detected.The positive samples are sequenced.After sequence splicing,online blast comparison is carried out on the website of national biological information center(NCBI),and phylogenetic analysis is carried out based on mega software.Confirm and identify the species of tick-borne protozoa infected or carried,and finally determine the type of pathogen infected by patients and host animals.4.Morphological microscopic examination:blood smears were prepared from the blood ofpeople and experimental animals.The blood smears prepared by Gimsa dye treatment were observed by optical microscope,and the infection of red blood cells in blood with protozoa was detected by fluorescence in situ hybridization(FISH),and the morphology,growth and development stage of protozoa were recorded.5.In vivo culture and identification of Babesia:inject positive blood samples into severe combined immunodeficiency(SCID)mice,regularly detect the erythrocyte infection of mice through PCR amplification and blood smear Giemsa staining,and observe whether Babesia is stably subcultured in mice.6.Follow up investigation of typical patients:carry out epidemiological investigation on typical patients infected with tick borne diseases,record the patient’s past history,epidemiological history and current medical history,carry out follow-up detection and observe their prognosis.Results1.Based on the surveillance survey of Babesia in the Northeast population of sentinel hospital:at the sentinel of Mudanjiang forestry Central Hospital,the blood samples of 507patients bitten by ticks and other patients in the epidemic area were collected.It was found that 3 people(0.59%)were infected with Babesia.After sequential comparative analysis,it was determined that they were infected with Babesia venatrum.The three patients from Heilongjiang province had no typical symptoms of recessive infection.2.The first discovery and confirmation of Babesia venatrum in Inner Mongolia:based on the fifth medical center of PLA General Hospital,Babesia was found in the blood of a patient from Inner Mongolia by screening patients with relevant clinical symptoms and eliminating other suspected diseases.Firstly,the blood smear was observed under the light microscope,and the suspected Babesia was found in the red blood cells;After that,the short fragment and full-length gene sequence of the 18S r RNA target gene of Babesia and a functional geneβ-tubulin gene sequence were obtained by PCR amplification.Through sequence alignment,it was determined that the patient was infected with Babesia venatrum.Human blood samples were inoculated with mice.On the 3rd,6th and 12th days after inoculation,the red blood cells of mice infected with Babesia were observed in the blood smears,but the PCR detection was negative.Three months later,the patient was followed up.A small amount of Babesia was still found in the patient’s blood through large-volume hemoconcentrated erythrocyte nucleic acid extraction and PCR amplification and FISH detection techniques.3.Investigation of protozoa equine,the host animal in Inner Mongolia,and the discovery ofa new type of Colpodella:in the blood samples of 400 horses collected in Inner Mongolia,136(34.0%)horses were detected to be infected with tick borne protozoa by PCR amplification and sequencing,of which 132(33%)horse blood samples were infected with Theileria equi.Two(0.5%)whole blood samples were infected with Babesia caballi and two(0.5%)whole blood samples were infected with Colpodella sp.Statistical analysis showed that the infection rates of the three pathogens in horses were significantly different(P<0.001,c~2=280.296).The homology of two Colpodella sequences with Colpodella sp.ATCC50594strain(Gen Bank No.AY14207583)was 99.18%and 98.73%respectively,and 89.23%and89.18%with Colpodella sp.HLJ strain(Gen Bank No.KT364261)from patients in Mudanjiang,Heilongjiang Province found by our research group five years ago.The homology with Colpodella sp.RRJ-2016 strain(Gen Bank No.KT600661)from Ixodes persulcatus in Mudanjiang is 88.54%and 88.42%respectively.It is a new type of Colpodella,and its pathogenicity and infectivity need to be further studied.4.Epidemiological investigation of new tick-borne protozoa in vector ticks in northern China:a total of 718 vector ticks were collected in Heilongjiang,Jilin,Inner Mongolia,Qinghai and other northern regions of China.Through morphological identification,there are7 species,including 101 Dermacentor nuttalli,24 Ixodes persulcatus,39 Dermacentor silvarum,72 Haemaphysalis concina,428 Haemaphysalis qinghaiensis,47 Dermacentor abaensis and 7 Ixodes ovatus.A total of 40 protozoa positive samples were detected,and the total positive rate was 5.57%.Sequence alignment analysis showed that there were abundant species of protozoa,including 3 Babesia(0.42%),34 Theileria(4.74%)and 3 Colpodella(0.42%).Among them,Babesia caballi was detected in Dermacentor nuttalli,and Theileria sinensis,Babesia sp.Irk-Ip525,Colpodella tetrahymenae,Uncultured Colpodellidae and Uncultured Colpodella were detected in five Haemaphysalis concinna.1 B.bigemina,19 T.uilenbergi,11 T.luwenshuni,1 Theileria sp.HU212 and 1 Theileria sp.LD188 were detected in Haemaphysalis qinghaiensis.1 T.luwenshuni was detected in Dermacentor abaensis.Conclusion1.This study further confirmed the invisible infection and natural epidemic area of Babesia venatrum in a high proportion of people in the key epidemic areas of tick-borne diseases in Northeast China,especially in asymptomatic patients,suggesting the risk of blood transfusion and donation safety of healthy people.2.It is the first time to confirm the infection case of Babesia venatrum in Inner Mongolia.The follow-up further confirmed the risk of difficult clearance of Babesia,suggesting the great importance of preventing transmission of protozoa.3.The full length and length of 18S r RNA gene of Babesia venatrum were obtained for the first time in human patientsβ-tubulin gene sequence enriches the genetic information resource database and understanding of the protozoa.4.It is the first time that horses,the host animals,have been found to be infected with the new type of Colpodella in Inner Mongolia.In view of the close relationship between horses and humans and as the blood sucking host of ticks,the role of horses in the transmission of this kind of protozoa is worthy of further monitoring and investigation.5.The species of new protozoa in vector ticks in northern China are complex and diverse.The risk of infection of new tick-borne protozoa diseases in northern China needs to be further investigated. |
