Molecular detection and identification of “Candidatus Ehrlichia hainanensis”, a novel Ehrlichia species in rodents from Hainan province, China

doi:10.21203/rs.3.rs-68850/v2

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Molecular detection and identification of “Candidatus Ehrlichia hainanensis”, a novel Ehrlichia species in rodents from Hainan province, China

https://doi.org/10.21203/rs.3.rs-68850/v2

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Background: Ehrlichia is a genus of Rickettsiales bacteria closely related to human/animal disease and it plays an important role in public health. Naturally they are hosted by mammals such as canines, bovines, wild rodents and vectored by ticks.

Results: In this study, we collected 121 rodent samples including 67 Niviventer fulvescens, 27 Rattus tanezumi, 24 Chiromyscus sp., 2 Rattus nitidus and 1 Leopoldamys edwardsi from Hainan province, which consists of the second largest island in China. The presence and genetic diversity of Ehrlichia was evaluated and characterized by amplifying and sequencing the 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 show highest 99.20%, 89.87% and 83.86% identity to known Ehrlichia sequences. In the phylogenetic trees they form a distinct cluster from any other species.

Conclusions: We propose that this species present a putative novel Ehrlichia species nominated as ‘Candidatus Ehrlichia hainanensis’. Its pathogenicity to human is still to be further researched and molecular surveillance in local populations is needed.

Parasitology

Niviventer fulvescens

Candidatus Ehrlichia hainanensis

Hainan province

Ehrlichia is a genus of obligatory intracellular bacteria belonging to family Anaplasmataceae, order Rickettsiales. Until today, six formally recognized Ehrlichia species [1] and some Candidatus species have been reported [2-6]. They are mainly vectored by ticks and many of them are pathogenic to human or animals. E. canis, E. ruminantium and E. muris are pathogens for canines, ruminants and mice, respectively [7-9]. E. chaffeensis, E. ewingii, E. muris-like agent and E. canis have been reported to infect human and cause syndromes ranging from febrile to severe multiple organ failure [10-14]. The common symptoms of human ehrlichiosis are fever, headache, myalgia, malaise, weakness, nausea, leukopenia, vomiting, diarrhea, and abdominal pain [15]. These uncharacteristic symptoms cause difficulties in diagnosis and may lead to misdiagnosis clinically. In China, multiple Ehrlichia species have been identified including E. chaffeensis, E. canis, E. ruminantium, etc, from ticks, mammals, mosquitoes and leeches [16-19] Until today, E. chaffeensis is the only known human Ehrlichial pathogen in China. Human granulocytic ehrlichiosis (HGE) disease caused by E. chaffeensis has been proved to be widespread in several provinces of China [16, 20-21]. Rural residents, especially farmers, are at substantially increased risk of Ehrlichia exposure [22].

Small mammals such as rodents have been proved to be the reservoirs of many Ehrlichia species. Ehrlichia sp. HF group has been identified in Apodemus argenteus, A. speciosus, Eothenomys smithi and Myodes rufocanus bedfordiae [23-25]. Candidatus E. khabarensis was identified and characterized from Myodes rutilus, M. rufocanus and Sorex araneus in Russian Far East [26]. E. muris was first isolated from the tissue of a wild mouse (Eothenomys kageus) in Japan [27], while a human pathogenic E. muris-like agent was identified from Peromyscus leucopus in US [28]. In the host-vector ecosystem, these Ehrlichia bacteria can be horizontally transmitted to ticks that infesting on the hosts, meanwhile, spillover into human populations may also occasionally occur. However, it is still unclear whether they pose a threat to public health. The solid evidence of their involvement in the human ehrlichiosis is still to be further studied.

Although the HGE has been common in China, the geographical distribution and genetic diversity of various Ehrlichia species have not yet been well studied. In Hainan Province, the investigations and studies on Ehrlichia have been very few [29]. Hainan province consists of the second largest island of China locating in Southern China Sea. It has an area 33, 900 square kilometers and a population of about 9.34 million. Owing to the tropical climate and outstanding landscape, it is becoming a great tourist attraction and is receiving tens of millions of tourists each year. For better understanding on the Ehrlichia distribution and its potential risk to public health in Hainan Province, we examined the molecular evidence of Ehrlichia in wild rodents and a novel Ehrlichia species was characterized in this study.

Sample collection and treatment

From November to December 2019, rodents were trapped by cages using bait. In total, 121 rodents were captured in Qiongzhong autonomous county, Hainan province. All animals were captured alive, and then anesthetized and euthanized to minimize suffering. The tissue samples were collected and stored in RNAlater. After washing twice with PBS, the liver samples were subjected to total DNA extraction using DNA/RNA isolation kit (Omega Bio-Tek, USA) according to the manufacturer’s instructions. The extracted DNA was stored at -20◦C prior to the species identification and Ehrlichia detection. The rodent species were identified by sophisticated field biologists and then confirmed by sequencing the cytochrome oxidase subunit 1 (COI) gene.

Ehrlichia detection and key genes amplification

Ehrlichia was detected using hemi-nested PCR primers targeting a conserved region of 16S rRNA gene as described [18]. The length of PCR fragments was 546 bp. Amplification parameters for both the first and second PCR included 94◦C×3min, followed by 40 cycles of: 94◦C×30 s, 49◦C×40 s, and 72◦C×45 s.

For better phylogenetic analysis, a 16S rRNA fragment of 1256 bp was obtained from the positive samples using primers as previously reported (18). The heat shock protein gene (groEL) fragments of 1116 bp in length were amplified using hemi-nested PCR primers [30]. The 970 bp citrate synthase gene (gltA) fragment was amplified using primers as described [30]. The PCR reactions were performed using Sensoquest PCR System LabCycler Standard P (Germany). PCR amplicons were analyzed by electrophoresis in 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 pMD19-T cloning vector (TaKaRa), transformed into E. coli and plated onto culture dish. The obtained clones were picked and sent for Sanger sequencing.

Phylogenetic analysis

Ehrlichia sequences (16S rRNA, groEL and gltA sequences) obtained in this study, as well as those retrieved from GenBank (Table S1), were aligned using the Clustal W method implemented in the MEGA program, version 6.0 [31]. Nucleotide sequence identities were calculated by MegAlign program available within the DNASTAR Lasergene package, version 7.0 (DNASTAR, Inc., Madison, WI).

Phylogenetic trees of basing on these three genes were constructed using the maximum likelihood (ML) method in the PhyML v3.0 package [32] based on the best-fit GTR+I+Γ4 model of nucleotide substitution as determined by jModeltest. Bootstrap values higher than 70% were considered significant. The best-fit evolutional model was determined using jModel Test version.

Detection of Ehrlichia in rodents and amplification of key genes

During 2019, a total of 121 rodents were captured in Qiongzhong autonomous county, Hainan province, Southern China (Fig. 1). These rodents represent 5 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 Ehrlichial DNA in 121 liver tissue samples. Consequently, PCR products of the expected size (546 nt) were recovered from 5 N. fulvescens (5/67, 7.46%). The result was confirmed by DNA sequencing. No positive result was detected from other rodent samples.

Genetic and phylogenetic analysis

Partial 16S rRNA (1256 bp), groEL (1116 bp) and gltA (970 bp) sequences were successfully obtained from 3 randomly selected samples. Basing on BLAST and the MegAlign analysis, the 16S gene sequences of the 3 strains have a nucleotide identity of 99.92-100% to each other, and they show highest 99.20% nucleotide identity with the Ehrlichia sp. EHt224 characterized from Hyalomma truncatum in Niger (AF311968.1) [33] and Ehrlichia sp. EBm52 from Rhipicephalus microplus in Thailand (AF497581.1) [34], respectively. The gltA gene shows highest 83.86% nucleotide identity to those from Ehrlichia sp. TC248-16 reported in Xinjiang [35], while highest 89.87% nucleotide identity with Ehrlichia sp. TC248-16 was identified for the groEL gene. In all the 3 phylogenetic trees, the sequences obtained in this study form a distinct cluster far from any other Ehrlichia species. We propose that it represents a novel species and we nominate it as Candidatus E. hainanensis. All the sequences have been submitted to Genbank (Accession numbers: MT875365-MT875373).

Rodents are the main reservoirs of a great many of human pathogens, and they play a key role in the transmission of zoonotic diseases such as hemorrhagic fever with renal syndrome (HFRS) [36], Lassa fever [37], rickettsiosis [38], etc. In this study, rodents including R. tanezumi, N. fulvescens, Chiromyscus sp., R. nitidus and L. edwardsi were captured from Hainan province were detected for the presence of Ehrlichia using hemi-nested PCR targeting the the 16S rRNA genes. Subsequently, a novel Ehrlichia species which is most closely related to Ehrlichia sp. strain Tibet genetically was identified from N. fulvescens samples. N. fulvescens belonging to genus Niviventer, family Muridae is a widespread rodent in the mountain and forest area, Southern China. Although rodents have been well recognized as the reservoir of Rickettsiales bacteria, studies on Rickettsiales bacterial pathogens from Niviventer have been very few until now. This might be the first report of Ehrlichia species identified and characterized from Niviventer rodents. Although N. fulvescens are mainly distributed in mountain and forest areas, the ongoing urbanization and deforestation makes more opportunities for rodents to contact human. The high positive rate (7.46%) in N. fulvescens may suggest the potential risk of infecting human. Besides, as a reservoir of Ehrlichia bacteria, the N. fulvescens might be involved in transmission cycles of Candidatus E. hainanensis in the nature because free-living rodents are common hosts of ticks and they have the potential to transmit the endosymbiotic bacteria to ticks. It is still to be determined that whether ticks act as vectors in the cycle, which may further increase the exposure risk of human.

In this study, we obtained the partial sequence of the 16S rRNA gene (1256 bp), the groEL gene (1116 bp), and the sequence of a gltA gene fragment (970 bp) from three Ehrlichia strains. In all the three phylogenetic trees, the Candidatus E. hainanensis significantly differ from other Ehrlichia bacteria and form a distinct clade. Genetic analysis indicates that the highest similarity of nucleotide sequences between Candidatus E. hainanensis and other Ehrlichia species is 99.20% for the 16S rRNA, 89.87% for the groEL gene and 83.86% for the gltA gene. These results of molecular-genetic analysis sufficiently support our proposition that Candidatus E. hainanensis can represent a putative new Ehrlichia species.

This study provided a better understanding of the genetic diversity of Ehrlichia species in rodents from Southern China. Ehrlichia is a group of endosymbiotic bacteria closely related to human and animal diseases [39]. Until today, E. chaffeensis is the most prevalent Ehrlichia infecting human. HGE cases caused by E. chaffeensis have been frequently reported in China [16, 20-21]. Besides, E. ruminantium as well as several closely related Ehrlichia species, E. ewingii, E. canis and E. muris-like agent have also been proved to infect human [10-14, 40]. Although some Ehrlichia species were characterized from ticks or vertebrate animals in recent years [6, 41-45], few studies have been carried out on their pathogenicity to human beings. In this study, due to the far relationship between Candidatus E. hainanensis and other Ehrlichia species, it is difficult to speculate whether it may infect human or not. Its pathogenicity to human beings is still to be further researched in this area.

This study discovered a novel Ehrlichia species in N. fulvescens from Hainan Island and we named it as Candidatus E. hainanensis. Its pathogenicity is to be studied and the high positive rate (7.46%) may suggest a potential threat to public health.

Human granulocytic ehrlichiosis: HGE, maximum likelihood: ML.

Acknowledgements

No applicable.

Funding

This work was funded by National Important Scientific & Technology Project (2018ZX10101002-002, 2018ZX10732401-001) and the National Natural Science Foundation of China (Grants 81702016).

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article or uploaded to GenBank.

Authors’ contributions

KL conceived the study. PL and HZ performed the molecular identification of pathogens and wrote the first draft of the manuscript. FX and YG worked in fields and collected the samples. ML performed the phylogenetic analysis. KL reviewed the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interest.

Consent for publication

Not applicable.

Ethics approval

This study was reviewed and approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention of the Chinese CDC.

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Molecular detection and identification of “Candidatus Ehrlichia hainanensis”, a novel Ehrlichia species in rodents from Hainan province, China

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