Distribution,Transmission Risk And Molecular Characterization Of Two Important Zoonotic Pathogens In Their Major Animal Hosts

Background: Leptospirosis is world-spreading infectious zoonoses caused by infection of pathogenic Leptospira species.China is also one of the main epidemic areas of leptospirosis.The incidence of human leptospirosis in China has significantly decreased in the recent decades.Since the great varieties of pathogenic Leptospira species and their host animals as well as long-time survival in natural water of the spirochetes that easily causes outbreak of disease,leptospirosis is still controlled as one of the national monitoring infectious diseases in China.Small mammalian animals act as the major reservoir hosts and transmission vectors of pathogenic Leptospira species,but the species,distribution and transmission risk of pathogenic Leptospira in small mammalian host animals remain poorly understood.The aim of this study is to investigate the species,distribution and transmission risk of pathogenic Leptospira in small mammalian host animals in central and eastern China and further understand the prevalence of pathogenic Leptospira species in small mammalian animals in our country to provide baseline data and scientific evidence for risk evaluation of leptospirosis outbreak.Methods: The renal specimens from 4001 small mammalian animals(1787 rats in 14 species,1790 bats in 20 species and 424 shrews in 4 species)captured in Hubei and Zhejiang provinces of China during November of 2016 to July of 2017 were collected to extract total DNAs.The universal 16 S RNA genes(rrs)of pathogenic Leptospira species in the total renal DNAs were detected by nested-PCR to determine the Leptospira-infected status in the animals.The sec Y,Lip L32,Lip L41,and fla B gene of pathogenic Leptospira species of rrs gene-positive DNA specimens were further detected by nested-PCRs for analysis of sequence homology and genetic phylogenesis to determine the main pathogenic Leptospira species in the animals.Results: The nested-PCR showed that 9.4%(375/4001)of the renal DNA specimens were positive for rrs gene sagments of pathogenic Leptospira species.The sequence homology and genetic phylogenesis analysis based on the rrs,sec Y,Lip L32,Lip L41,and fla B genes of pathogenic Leptospira species showed that the 375 positive small mammalian animals were infected with pathogenic L.interrogans,L.borgpetersenii,L.kirschneri and six potential novel pathogenic Leptospira species,respectively.All the potential novel pathogenic Leptospira species were detected in bats.The positive rate of L.interrogans in the rats was significantly higher than that in the bats or shrews while the positive rates of L.borgpetersenii in the rats or bats were significantly higher than that in the shrews and the positive rate of L.kirschneri in the bats was significantly higher than that in the rats or in shrews.Conclusion: The relatively high infection rate(9.4%)of pathogenic Leptospira species in the small mammalian hosts indicate a higher risk of huamn leptospirosis outbreak.L.interrogans,L.borgpetersenii and L.kirschneri are the predominant pathogenic Leptospira species infected in the small mammalian hosts.The bats carry several potential novel species of pathogenic Leptospira.As the dominant species of rats in mountainous area,Niviventer confucianus also is the major host of pathogenic Leptospira.In the small mammalian hosts,the rats are the main hosts of L.interrogans while the bats are the main hosts of L.borgpetersenii and L.kirschneri.Background: Tick-borne rickettsioses caused by tick-borne rickettsiae are the world-spreading infectious zoonoses.Ticks act as the main reservoir hosts and dissemination vectors of rickettsiae and play an important role in transmission of rickettsioses.Different geographic,geomorphic and climatic environments determine the distribution of different tick species and tick-borne rickettsial species.Many tick-borne rickettsiae have been isolated and identified from the mainland of China and most of them distribute in the areas of Northern China.In addition,the reported cases of rickettsiosis patients have been continuously increased in the recent years and most of them are from Northeastern China.However,the distribution of ticks and tick-borne rickettsial species in different areas of Northeastern China remain poorly understood.This study collected multiple species of ticks for detecting tick-borne rickettsiae to understand the distribution pattern and molecular characterization of the rickettsiae in ticks that provide scientific basis for more effective prevention and control of tick-borne rickettsioses in the area.Methods: The adult ticks were collected from the livestock,shrub and grasslands in seven regions with different geographic and geomorphic environments in Harbin area of Northeastern China during April to May of 2019.Species of the collected ticks were morphologically identified and then the mitochondrial 16 S r DNA genes of ticks were detected by PCR and sequencing for molecular biological identification and phylogenetic analysis.Rickettsia-specific citrogenase-encoding gene(glt A)and 17 k Da protein antigen-encoding gene(17-k Da)were detected by Nested-PCRs to screen the rickettsiae in the ticks.Several PCRs were also performed to amplify the long-segments of 16 S r RNA(rrs),glt A,gro EL and omp A genes and the amplification products were sequenced for analysis of sequence homology and genetic phylogenesis.Results: 1286 adult ticks belonging to five species were captured from the seven sampling regions of Harbin area.Haemaphysalis longicornis and Ixodes persulcatus are the predominant tick species in the livestock and shrub/grasslands,respectively.The nested-PCR showed that 370 of the 1286 ticks were detectable for rickettsial glt A and 17-k Da genes and the total rickettsial positive rate was 28.8%(370/1286).The sequencing data indicated that only two rickettsial species,Rickettsia raoultii and Candidatus Rickettsia tarasevichiae were detected from the rickettsial glt A and 17-k Da gene positive specimens.R.raoultii was found in the 37.6% of ticks belonging to D.nuttalli,D.silvarum and H.longicornis while Ca.R.tarasevichiae was presented in the 22.8% of ticks belonging to I.persulcatus alone.In particular,40.7% of the ticks from livestock were positive for both R.raoultii and Ca.R.tarasevichiae,which was significantly higher than that from the shrub/grasslands(19.5%)(P < 0.05).Conclusion: The ticks from Harbin area of Northern China present diversities of species and their distribution due to geography and geomorphy differences.R.raoultii and Ca.R.tarasevichiae are the two rickettsial species carried by the ticks.The higher carrying rate of the ticks from livestock indicates a tick-livestock rickettsial circulation that possibly play an important role in transmission of human rickettsioses.