Molecular Profiling Of Pathogenic Microorganisms In Bats From Mengyin, Shandong Province Of China

Background As the second largest mammalian group by a number of species,bats provide a large breeding ground for microorganisms.Bats are the only flying mammal,living socially with a large number in a diversity of niches worldwide.The ability to fly enables bats to carry microorganisms to distant areas.The worldwide distribution and inhabitation of various niches pose the public to a general risk of bat-borne microorganisms.Bats are social animals and the large number of bats in a colony with physical proximity facilitates the transmission of microorganisms among bats,making these microorganisms circulating stably among bats.Bats are ancient species with long life-span.The long evolutionary history provides long period for microorganisms to co-evolve with bats and makes bats their natural reservoirs.The longevity of bats contributes to the dispersal of microorganisms.During winter,bats hibernate to conserve energy.Their body temperature and metabolic rate are reduced,which may suppress immune responses and delay the clearance of microorganisms.Bats are frequently considered the reservoir host for a broad variety of newly emerging viruses,and some of these newly emerged viruses,such as SARS-Co V,MERS-Co V,Henipaviruses,Ebola virus and Marburg virus,are highly pathogenic to humans.Since the outbreak of SARS in 2003,there has been surging interests in bat-borne viruses,and a huge diversity of novel viruses were isolated or detected in bats.As bats do not develop severe clinical symptoms upon infection,they are considered as reservior hosts of these viruses.So far,the role of bats in the epidemiology and spillover of zoonotic diseases has not been fully understood,especially for batcterial and parasital agents.Objective To better understand the potential health risk of bats to humans,the viral,bacterial and parasital agents haborded by bats from Mengyin County,Shandong Province of China were investigated.Methods A total of 145 insectivorous bats were collected from Mengyin County,Shandong Province of China.Based on cyt B gene,bats were identified into six species of two bat families,Rhinolophus ferrumequinum and Rhinolophus pusillus in the family of Rhinolophidae;Eptesicus serotinus,Myotis fimbriatus,Myotis ricketti and Myotis pequinius in the family of Vespertilionidae.DNA/RNA was extracted from bat tissues and bloodsamples and screend for viral,bacterial and parasital agents using PCR with specific primers.1.Bat intenstine/lung samples were screened with degenerate primers for viruses of six families,including Coronaviruses,Astroviruses,Adenoviruses,Circoviruses,Hantaviruses and Paramyxoviruses.2.Bat bat blood samples were initially screened for the RNA polymerase B(rpo B)gene of Bartonella species.For rpo B positive samples,the citrate synthase(glt A)gene was amplifed for Bartonella genotypying.3.Bat spleen samples were screened for Borrelia by PCR targeting the 16 S rRNA(rrs2),glycerophosphodiester phosphodiesterase(glp Q)and flagellin(fla B)genes,respectively,which were commonly used for the characterization of relapsing fever Borrelia.4.To identify the species of Leptospira in bats,the rrs2 gene was amplified from bat kidney samples using nested PCR.To characterize the Leptospira species detected in bats from Mengyin,multilocus sequence typing(MLST)on 7 housekeeping genes(glm U,pnt A,suc A,tpi A,pfk B,mre A and cai B)was attempted.5.Blood samples were screened for Rickettsia by real-time PCR targeting the glt A and RC0338 genes.6.Blood samples were screened for Babesia by amplifying the 18 S r RNA and mitochondrial cytochrome C oxidase subunit ?(cox1)genes.7.Specific PCR targeting on the glycosomal glyceraldehyde phosphate dehydrogenase(g GAPDH)and 18 S r RNA genes were conducted to determine the diversity of trypanosomatids in bats from Mengyin.Results1.Bats from Mengyin were positive for Coronaviruses,Astroviruses,Adenoviruses and Circoviruses,and the overall infection rates for each virus were 21.4%(31/145),15.9%(23/145),20%(29/145)and 35.2%(51/145),respectively.Four bat species from the family of Vespertilionidae were all positive for Coronaviruses,Astroviruses,Adenoviruses and Circoviruses,while two species from the family of Rhinolophidae were both positive for Circoviruses,with one species positive for Coronaviruses and neither positive for Astroviruses and Adenoviruses.Hantaviruses and Paramyxoviruses were not found in bats from Mengyin.The highest positive rates for Astroviruses,Adenoviruses and Circoviruses were observed in Myotis fimbriatus and for Coronaviruses in Eptesicus serotinus.BLAST analysis showed that most of these virsues might be novel species due to their low nucletide and amino acid identities with the most closely known virus sequences.Based on DNA polymerase(pol)gene,Alphacoronavirus(?-Co V)and Betacoronavirus(?-Co V)were identified in bats from Mengyin.Interestingly,?-Co V identified in this study clustered with lineage C ?-Co V and shared85%–88% nucleotide identity with MERS-Co V.Based on the partial sequences of RNA-dependent RNA polymerase(Rd Rp)gene of Astroviruses,most of the bat Astroviruses detected in this study formed a separate bat Astroviruses lineage with previously described bat Astroviruses.However,several bat Astroviruses detected in this study were clustered with porcine/murine/rat Astroviruses.Based on the partial pol gene sequences,Adenoviruses detected in this study all belonged to the genus of Mastadenovirus.Phylogenetic analysis revealed that Adenoviruses detected in this study were host specificity,and there was possible cross-species transmission.Phylogenetic analysis of the partial replication-associated protein(Rep)gene sequences showed that circoviruses detected in this study showed no obvious host specificity and could be divided into 10 genotypes,with nine genotypes in the genus of Circovirus and one genotype in the genus of Cyclovirus.2.Bartonella was detected by PCR amplification of glt A gene in 25.2%(27/107)bats from Mengyin,including 1/3 Rhinolophus ferrumequinum,2/10 Rhinolophus pusillus,9/16 Myotis fimbriatus,1/5 Myotis ricketti and 14/58 Myotis pequinius.Phylogenetic analysis showed that Bartonella detected in bats from Mengyin clustered into ten groups,and some might represent novel species.There was an association between Bartonella species and bat species.In addition,co-infection with different Bartonella species in single bats was observed.3.Based on rrs2,glp Q and fla B genes,one Myotis ricketti was infected with a new Borrelia species(designated as Bat Borrelia sp.SD065),which was closely related to a cluster of New World Relapsing Fever Borrelia species including B.turicatae,B.parkeri and Candidatus B.johnsonii.4.By PCR amplifying of the rrs2 gene,50%(62/124)bats were infected with Leptospira,and Leptospira-positive bats were exclusively Myotis bats(Myotis fimbriatus,Myotis ricketti and Myotis pequinius).Leptospira identified in bats from Menying all belonged to the pathogenic group,including Leptpospira borgpetersenii,L.kirschneri,and several potentially new Leptospira species.Multilocus sequence typing defined three novel sequence types(STs)in L.kirschneri.Moreover,co-infection with different Leptospira species in bats from Mengyin was common.5.Genomic sequence for Rickettsia was not detected.6.Based on 18 S r RNA and cox1 genes,two Eptesicus serotinus were found positive for Babesia vesperuginis,which was pathogenic to bats.7.10.3%(13/126)bats(Eptesicus serotinus and Myotis pequinius)were positive fortrypanosomatid DNA,which were all identified as Trypanosoma dionisii.Conclusion1.Our study was the first to report the discovery of lineage C ?-Co V in Myotis fimbriatus and Eptesicus serotinus in China.This was also the first documentation of Coronaviruses in Myotis fimbriatus and Myotis pequinius,Adenoviruses in Eptesicus serotinus,Myotis fimbriatus and Myotis pequinius,expanding our understanding of the diversity of Coronaviruses and Adenoviruses in bats.2.This was the first study of Bartonella in bats from China,expanding our knowledge on the genetic diversity of Bartonella in bats,and sheding light on the ecology of bat-borne Bartonella species.3.The discovery of bat Borrelia sp.SD065 in China represented the first report of B.turicatae-like relapsing fever Borrelia in Old World,expanding our knowledge on the geographical distribution of New World Borrelia.4.Myotis bats in Mengyin showed a high infection rate with pathogenic Leptospira species,suggesting that Myotis bats were important carriers of Leptospira in Mengyin.5.Our study showed for the first time the presence of Babesia vesperuginis in Eptesicus serotinus collected from China,suggesting that Babesia vesperuginis had a broad host species and geographical distribution.6.Our study represented the first report of trypanosomatid parasites in bats from China,providing valuable information on the genetic diversity of these parasites in Asia.A diversity of potential novel viruses,bacteria and parasites were indetifed in bats from Mengyin.Although the pathogenesis of these microorganisms is unknown,monitoring of the potential spillover of bat-borne pathogenic microorgannisms to animals and humans is needed.