Discovery And Identification Of Human Infection Of Anaplasma Ovis

BackgroundsAnaplasma is a kind of intracellular parasitic bacteria.Anaplasmas reside in host blood cells and lead to the disease anaplasmosis.The pathogen can be transmitted to humans or animals by a variety of ticks,and is widespread in livestock.The genus Anaplasma includes Anaplasma phagocytophilum,A.capra,A.ovis,A.marginale,A.centrale,A.platys and A.bovis.The whole genome has been measured including A.marginale,A.centrale,A.phagocytophilum.There are three types of Anaplasma species that can be cultivated in vitro.A.phagocytophilum is likely to develop in both human cell lines(HL60)and tick cell lines(ISE6,IDE8).A.marginale can be successfully cultivated in the tick cell lines(IDE8,BME26).A.capra can isolate in culture in HL60.At present,it is thought three Anaplasma species have been identified as human pathogens.The first is A.phagocytophilum.In 1994,Americans first discovered that it could infect humans,and in 1996,Anaplasma were successfully isolated in culture from patients.Afterwards,human granulocytic anaplasmosis was covered a lot in the USA,Europe countries and China.Human granulocytic anaplasmosis can spread among human beings,and its common clinical symptoms include fever,headache,malaise,and myalgia.Common laboratory findings include leucopenia,thrombocytopenia,and high serum hepatic enzyme concentrations.The second agent of human anaplasmosis is an A.capra.In 2015,it was first reported that 28 patients were infected and the pathogen were isolated in vitro from the patients.The clinical manifestations were fever,headache,fatigue,dizziness and so on.In vivo target cells are not clear.The last agent of human anaplasmosis is an A.ovis variant.Up to now,only one Cypriot patient has been reported to be infected by the variety of A.ovis in diagnosed with 2007.In-vitro culture of A.ovis has not been reported by now.Mudanjiang area of Heilongjiang province and the Hengduan Mountains in Yunnan is China’s two important natural foci of tick borne disease.Previous investigations have shown that ticks and host animals of these regions carry a variety of tick borne pathogens,which is a key area of surveillance.It is thought that A.ovis lead to only mild clinical symptoms,so there is lack of systematic research,especially the identification and isolation for a long time.ObjectivesInvestigate the epidemiological characteristics of A.ovis in the ticks and livestocks to estimate the health risks to the local people.A.ovis was isolated from livestock,which provided the conditions to prepare antigen slides to confirm any infection with this bacterial.In order to find out the A.ovis cases,identify the pathogenic characteristics and describe the clinical manifestations,we carry out the surveillance of A.ovis infected in the sentinel hospital.MethodsFrom 2014 to 2016,every year in April to August ticks and host animal samples were collected in Mudanjiang area and Hengduan Mountains.Ticks and EDTA Blood samples were tested by polymerase chain reaction(PCR)for A.ovis.Investigate of natural foci of A.ovis pathogens to find out its epidemiologic features.Cultivation of A.ovis in the human promyelocytic leukemia(HL-60)cell line.PCR,IFA,FISH,Wright Giemsa staining and scanning electron microscope were indicated wether the HL-60 cells were infceted with A.ovis or not.Carry out active surveillance in sentinel hospitals.Patient had a history of tick bites were enrolled and through the questionnaire to obtain patient epidemiological information.Inpatient information is registered through the medical records.In this study,PCR was used to screen the patients,and IFA was used to diagnose the disease.FindingsThe investigation on natural foci,we found that six ticks were infected with A.ovis including Dermacentor silvarum 4(4/275),Ixodes persulcatus 1(1/990),Haemaphysalis concinna 1(1/239).The infection rate of A.ovis was 0.36%(6/1657).And the infection ratio of A.ovis was 22.39%(150/670)in livestock,12.10%(53/438)and 41.81%(97/232)in Mudanjiang area and Hengduan Mountains respectively.Successfully cultivation of A.ovis in HL-60 cell lines.PCR,IFA,FISH,Wright Giemsa staining and scanning electron microscope were indicated that the HL-60 cells were infceted with A.ovis.In 2014-2016,a total of 1631 blood samples were gathered from sentinel hospitals.386 paired sera of tick-bite patients were collected from 2010-2016.The IgG antibody of A.ovis were identified by IFA using serums from PCR positive patients and all 386 paired sera.1 patient with A.ovis infected was found in the active surveillance and retrospective investigation respectively.ConclusionAfter the investigation of the A.ovis vector ticks and host animals,we found that Dermacentor silvarum is the main vector of A.ovis in Mudanjiang,and such animals as the sheep,goats and cattle are the host animals of A.ovis.Infected ticks and livestock confirmed Mudanjiang area is the natural foci of A.ovis.A.ovis first in the world were successfully cultivated in HL-60 cells and clearly distinguished from other Anaplasma species.Based on the active surveillance,we found one case infected with A.ovis,which added the direct evidence that A.ovis can infect human-being.Significance and innovationDue to the mild symptoms of infection,we often ignore the the disease caused by the A.ovis.This study shows that the existence of A.ovis in livestock and the potential threat to human beings.A.ovis should be given enough attention.It is the first time to realize the isolation and culture of A.ovis from animal origin.Although A.ovis isolate in HL-60 cells could only be established for around four months,we can use this cultivation to prepare antigen slides to confirm any infection with this bacterial.The discovery of clinical infection cases provides an important basis of the further research of pathogenicity and clinical symptoms of this disease.