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ZHAO Hong Qing, LIU Pei Pei, XUE Feng, LU Miao, QIN Xin Cheng, LI Kun. Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China[J]. Biomedical and Environmental Sciences, 2021, 34(12): 1020-1023. doi: 10.3967/bes2021.138
Citation: ZHAO Hong Qing, LIU Pei Pei, XUE Feng, LU Miao, QIN Xin Cheng, LI Kun. Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China[J]. Biomedical and Environmental Sciences, 2021, 34(12): 1020-1023. doi: 10.3967/bes2021.138

Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China

doi: 10.3967/bes2021.138
Funds:  This work was funded by the National Important Scientific & Technology Project [2018ZX10101002-002 and 2018ZX10732401-001]; the Inner Mongolia Natural Science Foundation Project [grant No. 2016MS0859]; the Key Scientific and Technology Project of Inner Mongolia Autonomous Region [grant No. 2021ZD0006]; and the National Natural Science Foundation of China [grant No. 82102390]
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  • Author Bio:

    ZHAO Hong Qing, male, born in 1982, Doctor, Assistant Professor, majoring in infectious disease control

    LIU Pei Pei, male, born in 1985, Doctor, Associate Professor, majoring in zoonotic pathogens

  • Corresponding author: LI Kun, Tel: 86-10-58900783, Fax: 86-10-58900700, E-mail: likun@icdc.cn
  • &These authors contributed equally to this work.
  • Received Date: 2021-05-28
  • Accepted Date: 2021-09-21
  • Ehrlichia (Anaplasmataceae family) are obligatory intracellular bacteria that infect humans and animals. They are hosted by mammals such as canines, bovines and wild rodents, and are vectored by ticks. In this study, we collected 121 rodent samples comprising 67 Niviventer fulvescens, 27 Rattus tanezumi, 24 Chiromyscus sp., 2 Rattus nitidus and 1 Leopoldamys edwardsi from Hainan province, which includes the second largest island in China. The presence and genetic diversity of Ehrlichia species was evaluated and characterized by amplification and sequencing of 16S rRNA, groEL and gltA genes. An Ehrlichia species was detected in 5 of the 67 Niviventer fulvescens samples (7.46%). The 16S rRNA, groEL and gltA genes showed the highest identity to known Ehrlichia sequences (99.20%, 89.87% and 83.86%, respectively). In the phylogenetic trees they formed a cluster distinct from all other species. We propose that this species is a putative novel Ehrlichia species, which we suggest be named Candidatus Ehrlichia hainanensis. Its pathogenicity to humans remains to be further researched, and molecular surveillance in local populations is needed.
  • &These authors contributed equally to this work.
  • 加载中
  • [1] Parte AC. LPSN - list of prokaryotic names with standing in nomenclature (bacterio net), 20 years on. Int J Syst Evol Microbiol, 2018; 68, 1825−9. doi:  10.1099/ijsem.0.002786
    [2] Dumler JS, Bakken JS. Ehrlichial diseases of humans: emerging tick-borne infections. Clin Infect Dis, 1995; 20, 1102−10. doi:  10.1093/clinids/20.5.1102
    [3] Parzy D, Davoust B, Bissuel G, et al. Human pathogenicity of Ehrlichia canis. Lancet, 1991; 337, 1169.
    [4] Buller RS, Arens M, Hmiel SP, et al. Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis. N Engl J Med, 1999; 341, 148−55. doi:  10.1056/NEJM199907153410303
    [5] Johnson DKH, Schiffman E, Davis JP, et al. Human infection with Ehrlichia muris-like pathogen, United States, 2007-2013. Emerg Infect Dis, 2015; 21, 1794−9. doi:  10.3201/eid2110.150143
    [6] Olano JP, Masters E, Hogrefe W, et al. Human monocytotropic ehrlichiosis, Missouri. Emerg Infect Dis, 2003; 9, 1579−86. doi:  10.3201/eid0912.020733
    [7] Guo WP, Tian JH, Lin XD, et al. Extensive genetic diversity of Rickettsiales bacteria in multiple mosquito species. Sci Rep, 2016; 6, 38770. doi:  10.1038/srep38770
    [8] Zhang XC, Zhang LX, Li WH, et al. Ehrlichiosis and zoonotic anaplasmosis in suburban areas of Beijing, China. Vector Borne Zoonotic Dis, 2012; 12, 932−7. doi:  10.1089/vbz.2012.0961
    [9] Lu M, Tian JH, Yu B, et al. Extensive diversity of rickettsiales bacteria in ticks from Wuhan, China. Ticks Tick Borne Dis, 2017; 8, 574−80. doi:  10.1016/j.ttbdis.2017.03.006
    [10] Saito TB, Walker DH. Ehrlichioses: an important one health opportunity. Vet Sci, 2016; 3, 20. doi:  10.3390/vetsci3030020
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Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China

doi: 10.3967/bes2021.138
Funds:  This work was funded by the National Important Scientific & Technology Project [2018ZX10101002-002 and 2018ZX10732401-001]; the Inner Mongolia Natural Science Foundation Project [grant No. 2016MS0859]; the Key Scientific and Technology Project of Inner Mongolia Autonomous Region [grant No. 2021ZD0006]; and the National Natural Science Foundation of China [grant No. 82102390]
  • Author Bio:

  • Corresponding author: LI Kun, Tel: 86-10-58900783, Fax: 86-10-58900700, E-mail: likun@icdc.cn
  • &These authors contributed equally to this work.

Abstract: Ehrlichia (Anaplasmataceae family) are obligatory intracellular bacteria that infect humans and animals. They are hosted by mammals such as canines, bovines and wild rodents, and are vectored by ticks. In this study, we collected 121 rodent samples comprising 67 Niviventer fulvescens, 27 Rattus tanezumi, 24 Chiromyscus sp., 2 Rattus nitidus and 1 Leopoldamys edwardsi from Hainan province, which includes the second largest island in China. The presence and genetic diversity of Ehrlichia species was evaluated and characterized by amplification and sequencing of 16S rRNA, groEL and gltA genes. An Ehrlichia species was detected in 5 of the 67 Niviventer fulvescens samples (7.46%). The 16S rRNA, groEL and gltA genes showed the highest identity to known Ehrlichia sequences (99.20%, 89.87% and 83.86%, respectively). In the phylogenetic trees they formed a cluster distinct from all other species. We propose that this species is a putative novel Ehrlichia species, which we suggest be named Candidatus Ehrlichia hainanensis. Its pathogenicity to humans remains to be further researched, and molecular surveillance in local populations is needed.

&These authors contributed equally to this work.
ZHAO Hong Qing, LIU Pei Pei, XUE Feng, LU Miao, QIN Xin Cheng, LI Kun. Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China[J]. Biomedical and Environmental Sciences, 2021, 34(12): 1020-1023. doi: 10.3967/bes2021.138
Citation: ZHAO Hong Qing, LIU Pei Pei, XUE Feng, LU Miao, QIN Xin Cheng, LI Kun. Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China[J]. Biomedical and Environmental Sciences, 2021, 34(12): 1020-1023. doi: 10.3967/bes2021.138
  • Ehrlichia is a genus of obligatory intracellular bacteria belonging to the family Anaplasmataceae, order Rickettsiales. To date, six formally recognized Ehrlichia species and some Candidatus species have been reported[1]. They are mainly vectored by ticks, and many are pathogenic to humans and animals. Ehrlichia canis (E. canis), Ehrlichia ruminantium (E. ruminantium) and Ehrlichia muris are pathogens for canines, ruminants and mice, respectively. Ehrlichia chaffeensis (E. chaffeensis), Ehrlichia ewingii, Ehrlichia muris-like agent and E. canis have been reported to infect humans and to cause symptoms ranging from fever to severe multiple organ failure[2-5]. The common symptoms of human ehrlichiosis are fever, headache, myalgia, malaise, weakness, nausea, leukopenia, vomiting, diarrhea and abdominal pain[6]. These indistinguishable symptoms cause difficulties in diagnosis and may lead to clinical misdiagnosis. In China, multiple Ehrlichia species have been identified, including E. chaffeensis, E. canis and E. ruminantium, from ticks, mammals, mosquitoes and leeches[7]. E. chaffeensis and E. canis are currently the known human Ehrlichia pathogens in China. Human monocytic ehrlichiosis disease caused by E. chaffeensis has been demonstrated to be widespread in several provinces of China[8]. Rural residents, particularly farmers, are at substantially increased risk of Ehrlichia exposure.

    Small mammals such as rodents have been demonstrated to be the reservoirs of many Ehrlichia species. The Ehrlichia sp. HF group has been identified in Apodemus argenteus, A. speciosus, Eothenomys smithi and Myodes rufocanus bedfordiae. Candidatus E. khabarensis was identified and characterized from Myodes rutilus, M. rufocanus and Sorex araneus in the Russian Far East. Ehrlichia muris was first isolated from the tissue of a wild mouse (Eothenomys kageus) in Japan, and a human pathogenic E. muris-like agent was identified from Peromyscus leucopus in the United States. In the host-vector ecosystem, these Ehrlichia bacteria can be horizontally transmitted to ticks that infest hosts, and spillover into human populations may also occasionally occur. However, whether they pose a threat to public health remains unclear. Evidence of their involvement in the human ehrlichiosis remains to be further determined.

    Although human monocytic ehrlichiosis has been common in China, the geographical distribution and genetic diversity of various Ehrlichia species have not yet been well studied. In Hainan Province, few investigations and studies have been performed on Ehrlichia. Hainan Province contains the second largest island in China, located in the South China Sea. It has an area of 33,900 square kilometers and a population of approximately 9.34 million people. Owing to its tropical climate and landscape, it has become a major tourist attraction receiving tens of millions of visitors each year. To date, more than 20 tick species have been reported in Hainan, including Ixodes, Rhinpicephalus, Dermacentor, Amblyomma and Haemaphysalis. Vector surveillance has indicated that Hainan Province has the highest rat density in China. Multiple rodent species have been observed, including Niviventer fulvescens, Niviventer niviventer, Rattus rattus, Rattus norvegicus and Rattus flavipectus. To improve understanding of the Ehrlichia distribution in these vectors/hosts and the potential risk to public health in Hainan Province, we examined the molecular evidence of Ehrlichia in wild rodents and performed further research in this study.

    From November to December of 2019, rodents were trapped in cages by using bait. In total, 121 rodents were captured in Qiongzhong autonomous county in the middle of Hainan Province. All animals were captured alive and then anesthetized to minimize suffering. The rodents were sacrificed by cervical dislocation. The tissue samples were collected and stored in RNAlater. After being washed twice with phosphate buffer, the liver samples were subjected to total DNA extraction with a DNA/RNA isolation kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. The extracted DNA was stored at −20 °C before species identification and Ehrlichia detection. The rodent species were identified by experienced field biologists and then confirmed by sequencing of the mt-cyt b gene.

    Ehrlichia was detected with hemi-nested PCR primers targeting a conserved region of 16S rRNA as previously described[7]. The length of PCR fragments was 546 bp. The amplification parameters for both the first and second PCR were 94 °C for 3 min, followed by 40 cycles of: 94 °C for 30 s, 49 °C for 40 s, 72 °C for 45 s and an additional 10 min at 72 °C. For better phylogenetic analysis, a 16S rRNA fragment of 1,256 bp was obtained from the positive samples by using previously reported primers[7]. Heat shock protein gene (groEL) fragments 1,116 bp in length were amplified by conventional PCR with hemi-nested PCR primers[7]. The 970 bp citrate synthase gene (gltA) fragment was amplified with primers as previously described[7]. The PCR reactions were performed with a Sensoquest PCR System LabCycler Standard P (Germany). Amplification parameters for the first and second PCR included 94 °C for 3 min, followed by 40 cycles of: 94 °C for 30 s, 47 °C for 70 s, 72 °C for 45 s and an additional 10 min at 72 °C. PCR amplicons were analyzed by electrophoresis on 1.0% agarose gels. The PCR products shorter than 800 bp were subjected to Sanger sequencing by Sangon Biotechnology Company (Shanghai, China). The amplicons longer than 800 bp were cloned into the pMD19-T cloning vector (TaKaRa), transformed into E. coli and plated onto culture dishes. The obtained clones were collected and sent for Sanger sequencing.

    Ehrlichia sequences (16S rRNA, groEL and gltA sequences) obtained in this study, as well as those retrieved from GenBank (Supplementary Table S1 available in www.besjournal.com), were aligned with the Clustal W method implemented in the MEGA program, version 6.0[9]. Nucleotide sequence identities were calculated with the MegAlign program available in the DNASTAR Lasergene package, version 7.0 (DNASTAR, Inc., Madison, WI, USA). Phylogenetic trees based on these three genes were constructed with the maximum likelihood method in the PhyML v3.0 package (http://www.atgc-montpellier.fr/phyml/binaries.php) with the best-fit GTR+I+Γ4 model of nucleotide substitution, as determined by jModeltest. Bootstrap values higher than 70% were considered significant.

    No. GeneGenbank numberBacterial strain
    116SAY135531.1Ehrlichia sp. 'Rattus strain'
    216SAY098730.1Ehrlichia sp. Belluno
    316SKR063138.1Candidatus Ehrlichia khabarensis strain m3
    416SAB074459.1Candidatus Ehrlichia shimanensis
    516SKY425523.1Candidatus Ehrlichia sp. AG-2017 isolate Y272
    616SNR074513.2Ehrlichia ruminantium strain Welgevonden
    716SDQ324367.1Ehrlichia sp. P-Mtn
    816SCP000236.1Ehrlichia chaffeensis str. Arkansas
    916SKX505292.1Ehrlichia chaffeensis isolate X1
    1016SAB454074.2Ehrlichia sp. NS101
    1116SNR121714.1Ehrlichia muris AS145
    1216SCP007474.1Ehrlichia sp. HF
    1316SGU227701.1Ehrlichia sp. Yunnan
    1416SNR044747.1Ehrlichia ewingii strain Stillwater
    1516SKF728345.1Uncultured Ehrlichia sp. clone SY36
    1616SAF311968.1Ehrlichia sp. EHt224
    1716SAF414399.1Ehrlichia sp. Tibet
    1816SAF497581.1Ehrlichia sp. EBm52
    1916SKM995821.1Uncultured Ehrlichia sp. clone Tajikistan-4
    2016SDQ324547.1Ehrlichia sp. Fujian
    2116SJX402605.1Uncultured Ehrlichia sp. clone Xinjiang158-10
    2216SKF843826.1Candidatus Ehrlichia regneryi clone Camel_17
    2316SAF318946.1Ehrlichia ovina
    2416SU26740.1Ehrlichia canis
    2516SAB723708.1Ehrlichia canis isolate W-134
    26gltADQ513396.1Ehrlichia ruminantium strain Senega
    27gltAKR063140.1Candidatus Ehrlichia khabarensis strain m3
    28gltAJX629807.1Ehrlichia minasensis strain UFMG-EV
    29gltAAY647155.1Ehrlichia canis
    30gltAKP719095.1Ehrlichia ovina isolate T2002
    31gltAAF304142.1Ehrlichia chaffeensis Arkansas
    32gltADQ647319.1Ehrlichia sp. HF
    33gltAMN685601.1Ehrlichia muris
    34gltAAF311966.1Ehrlichia sp. EHt224
    35gltAKX987356.1Ehrlichia sp. strain WHBMXZ-40
    36gltAJX402606.1Uncultured Ehrlichia sp. clone Xinjiang116-7
    37gltAKJ410275.1Ehrlichia sp. TC248-16
    38groELKJ930193.1Uncultured Ehrlichia sp. clone Tajikistan-3
    39groELJX402613.1Uncultured Ehrlichia sp. clone Xinjiang157-6
    40groELKY705065.1Ehrlichia sp. isolate YNT
    41groELAF195273.1Ehrlichia ewingii
    42groELKJ410296.1Ehrlichia sp. TC251-2
    43groELDQ647010.1Ehrlichia ruminantium strain Mara87/7
    44groELAB074462.1Candidatus Ehrlichia shimanensis
    45groELFJ966353.1Candidatus Ehrlichia khabarensis isolate Khabarovsk 1931
    46groELU96731.1Ehrlichia canis
    47groELJX629806.1Ehrlichia minasensis strain UFMG-EV
    48groELDQ672553.1Candidatus Ehrlichia ovata
    49groELKU214846.1Ehrlichia sp. EMLA
    50groELMN685610.1Ehrlichia muris

    Table S1.  Genbank numbers of bacterial sequences used in phylogenetic analysis

    In this study, a total of 121 rodents were captured in Qiongzhong autonomous county, Hainan Province, Southern China. These rodents represented five species: 67 Niviventer fulvescens, 27 Rattus tanezumi, 24 Chiromyscus sp., 2 Rattus nitidus and 1 Leopoldamys edwardsi. Hemi-nested PCR targeting the 16S rRNA gene was performed to screen the Ehrlichia DNA in 121 liver tissue samples. Consequently, PCR products of the expected size (546 nt) were recovered from five N. fulvescens (5/67, 7.46%). The results were confirmed by DNA sequencing. No positive results were detected from R. tanezumi and other rodent samples.

    Partial 16S rRNA (1,256 bp), groEL (1,116 bp) and gltA (970 bp) sequences were successfully obtained from three randomly selected samples. Through BLAST and MegAlign analysis, the 16S gene sequences of the three strains were found to have 99.92%–100% nucleotide identity, and the highest nucleotide identity (99.20%) was observed with the Ehrlichia sp. EHt224 characterized from Hyalomma truncatum in Niger (AF311968.1) and Ehrlichia sp. EBm52 from Boophilus microplus in Thailand (AF497581.1). The gltA gene showed the highest nucleotide identity to Ehrlichia sp. TC248–16 reported in Xinjiang (83.86%), whereas the highest nucleotide identity with Ehrlichia sp. TC248–16 (89.87%) was identified for the groEL gene. In all phylogenetic trees, the sequences obtained in this study formed a distinct cluster far from any other Ehrlichia species (Figure 1). We propose that this is a novel species, which we suggest be named Candidatus E. hainanensis. All sequences have been submitted to GenBank (MT875365–MT875373).

    Figure 1.  Phylogenetic tree based on the nucleotide sequences of Candidatus Ehrlichia hainanensis 16S rRNA (A), groEL (B) and gltA (C) genes, as well as those obtained from GenBank.

    Rodents are the main reservoirs of many human pathogens, and they play key roles in the transmission of zoonotic diseases, such as hemorrhagic fever with renal syndrome, Lassa fever and rickettsiosis. In this study, rodents including R. tanezumi, N. fulvescens, Chiromyscus sp., R. nitidus and L. edwardsi captured from Hainan Province were detected for the presence of Ehrlichia through hemi-nested PCR targeting the 16S rRNA genes. Subsequently, a novel Ehrlichia species genetically most closely associated with Ehrlichia sp. strain Tibet was identified from N. fulvescens samples belonging to the genus Niviventer. The family Muridae is widespread in mountain and forest areas in Southern China. Although rodents have been well recognized as the reservoir of Rickettsiales bacteria, very few studies on Rickettsiales bacterial pathogens from Niviventer have been performed to date. This is one of the few Ehrlichia species identified and characterized from Niviventer rodents. Although N. fulvescens are mainly distributed in mountain and forest areas, ongoing urbanization and deforestation provide increasing opportunities for rodents to contact humans. The high N. fulvescens positivity rate (7.46%) may suggest the potential risk of human infection. Furthermore, as a reservoir of Ehrlichia bacteria, N. fulvescens might be involved in transmission cycles of Candidatus E. hainanensis in nature, because free-living rodents are common hosts of ticks and have the potential to transmit Ehrlichia bacteria to ticks. Whether ticks act as vectors in the cycle remains to be determined; if so, the exposure risk to humans may be further increased.

    In this study, we obtained the partial sequence of the 16S rRNA gene (1,256 bp), the groEL gene (1,116 bp) and a gltA gene fragment (970 bp) from three Ehrlichia strains. In all three phylogenetic trees, the Candidatus E. hainanensis significantly differed from other Ehrlichia bacteria and formed a distinct clade. Genetic analysis indicated that the highest similarity of nucleotide sequences between Candidatus E. hainanensis and other Ehrlichia species was 99.20% for 16S rRNA, 89.87% for the groEL gene and 83.86% for the gltA gene. These results of molecular-genetic analysis sufficiently supported that Candidatus E. hainanensis is a putative new Ehrlichia species.

    This study provides a better understanding of the genetic diversity of Ehrlichia species in rodents from Southern China. Ehrlichia is a group of endosymbiotic bacteria that infect humans and animals. E. chaffeensis is currently the most prevalent Ehrlichia infecting humans. Human monocytic ehrlichiosis cases caused by E. chaffeensis have been frequently reported in China. Furthermore, E. ruminantium as well as several closely related Ehrlichia species, E. ewingii, E. canis and E. muris-like agent have also been demonstrated to infect humans[2-5, 10]. Although other Ehrlichia species have been characterized from ticks or animals in recent years, few studies have been performed on their pathogenicity to humans. In this study, owing to the distant relationship between Candidatus E. hainanensis and other Ehrlichia species, it is difficult to speculate whether this species might infect humans, and its pathogenicity to humans remains to be further researched in this area.

    Acknowledgements We sincerely thank Mrs. SUN Nan, Mrs. WANG Wen, and Mr. WANG Chang Shun for their kind help.

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