Co-infection of Anaplasma and Ehrlichia in Hedgehogs from China.

Hedgehogs ( Erinaceus amurensis ) is an insectivorous mammal frequently observed in the wild and around the residential areas. However, information about tick-borne diseases in this species is not well known. We investigated tick-borne rickettsial pathogens including Anaplasma , Ehrlichia, Candidatus . Neoehrlichia and Rickettsia in hedgehogs collected in central China. Hedgehogs were captured from Hubei and Jiangxi Province with living traps. PCR amplication and DNA sequencing showed that 26% (19/73) hedgehogs were positive to Anaplasma bovis, 20.5% (15/73) were positive to a tentative new Ehrlichia species; in addition, 13.7% (10/73) hedgehogs were positive to A. bovis and Ehrlichia simultaneously. Ca . Neoehrlichia and Rickettsia species was not detected among the 73 hedgehogs. We concluded that hedgehogs from central China were widely infected with Anaplasma and Ehrlichia , suggesting hedgehogs may play a role in the ecology of Anaplasma and Ehrlichia .


Background
Bacteria of the order Rickettsiales (Alphaproteobacteria) are obligate intracellular parasites of eukaryotes. At present, the order contains three established families (Rickettsiaceae, Holosporaceae, and Anaplasmataceae) as well as one proposed family (Candidatus Midichloriaceae) [1,2]. Most described species of Rickettsiales are well known as emerging or reemerging zoonotic pathogens that may cause life-threatening diseases, including rickettsioses, ehrlichiosis, anaplasmosis and scrub typhus in humans and also be linked with devastating agricultural losses by infecting meat and milk producing animals [3,4]. The number of newly discovered organisms in the Rickettsiales has markedly increased over the last 20 years and even bacteria that had previously been considered nonpathogenic are now associated with human disease [5,6]. Rickettsiales are associated with a diverse host range, including diverse protists, annelids, arthropods, mammals and birds [5,7,8]. Although numerous studies conducted worldwide have focused on the ecology of Rickettsiales, investigation related to vector-pathogen interaction and the role of vertebrate hosts in the maintenance and dissemination of Rickettsiales in nature remain scarce [9].
Previous investigations showed that ectoparasites in hedgehogs carried several rickettsial agents [10,11]. Rickettsia helvetica, Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum were also detected in European hedgehogs (Erinaceus europaeus) in Europe [10,[12][13][14]. Erinaceus europaeus has also been suggested as reservoir hosts for A.phagocytophilum and different genospecies of the Borrelia burgdorferi sensu lato complex [15,16]. Thus, hedgehog may play an important role in the transmission cycle of Rickettsiale species as well as acting as a reservoir. The aim of this study was to investigate the prevalence of Rickettsia, Anaplasma and Ehrlichia in hedgehogs from central China.

Results
Anaplasma in hedgehogs PCR ampli cation with Anaplasma rrs primers showed that 26% (19/73) hedgehogs were positive. DNA sequences analysis revealed that the sequences were 98.6-100% homologous to each other and they were 98.6-100% identical to A.bovis sequences derived from the blood of cattle and goat in Shaanxi Province, China (GenBank: MH255934 and MH255938). Phylogenetic analysis of two representative rrs sequences showed sequences from hedgehogs were in the same cluster with A. bovis detected in China, Japan, Korea and Malaysia (Fig.1). Additionally, all Anaplasma rrs positive samples were further genotyped using semi-nested PCR by targeting the groEL gene. A total of 18 highly identical (97.3%-100%) groEL sequences were nally obtained. BLAST analysis indicated all sequences were 97.86%-99.64% identical to the partial groEL sequence of A.bovis derived from a goat in Shaanxi Province (GenBank: MH255898). Phylogenetic analysis showed two representative groEL sequences obtained in the present study clustered with A.bovis detected in goats/cattle/ticks/mosquitos from Shaanxi and Hubei Province. They clearly formed a distinct clade and differ from A.bovis in other parts of China and the world (Fig.1).

Ehrlichia in hedgehogs
PCR ampli cation with Ehrlichia rrs primers showed that 20.5% (15/73) hedgehogs were positive. The 15 rrs positive hedgehogs were further ampli ed for the groEL and gltA genes. All 15 hedgehogs were positive to the groEL, but only 4 were gltA positive. BLAST analysis showed that the rrs sequences from hedgehogs were 99.13-99.35% identical to an Ehrlichia sequence (GenBank: KJ410252) derived from ticks in Xinjiang Province in western China. The four gltA sequences were 99.5-100% homologous and the groEL sequences detected from 15 hedgehogs were 99.1-100% identical to each other. In consistence with the rrs sequences, the gltA and groEL were highly homologous to Ehrlichia detected in ticks from various places in China including Xinjiang, Zhejiang, and Hubei in China, and E. ewingii (93.79%-95.04% for groEL and 91%-91.47% for gltA). Phylogenetic analysis based on the rrs showed that Ehrlichia sequences detected from hedgehogs formed a clade together with uncultured Ehrlichia species from ticks in Daishan County, Xinjiang provinces and E. ewingii (Fig.2). Phylogenetic analysis of groEL and gltA genes also showed that Ehrlichia detected in hedgehogs clustered together with uncultured Ehrlichia that was previously reported in ticks in Wuhan City, Xinjiang and Zhejiang provinces of China, and E. ewingii (Fig.3). The results indicated that the Ehrlichia from hedgehogs appeared to be a tentative new species which is taxonomically closely related to E. ewingii.

The infection rates of hedgehogs
The overall infection rate of A.bovis and Ehrlichia in hedgehogs was 26% and 20.5%, respectively. Coinfections were found in 10 hedgehogs (13.7%) ( Table 2). No genomic DNA of Ca. Neoehrlichia and Rickettsia was detected in our survey.
Histopathological examination of a road-killed hedgehog A seemingly intact but seriously road-injured adult hedgehog was found near a collection site in Wuhan city. The animal was later dead and dissected, tissue samples from liver, lung, kidney and spleen were xed in 10% formalin for further analysis and subsequent use (Fig.4). After 72 h, the samples were dehydrated, embedded in para n and cut into 5-mm thickness and strained with hematoxylin & eosin (H&E) (Fig.5).The animal was later molecularly screened positive both for Anaplasma and Ehrlichia.

Discussion
To the best of our knowledge, this is the rst identi cation of A. bovis and a tentative new Ehrlichia sp. in peripheral blood of hedgehogs. These results suggest that hedgehogs could play a role in the circulation of A. bovis and this tentative new Ehrlichia species. In several studies, Candidatus Neoehrlichia mikurensis were identi ed in mosquitoes, rodents and humans in China [9,17]. Rickettsia japonica is widely distributed in China [18,19]. We tested the primers for Rickettsia by PCR with positive R. japonica sample and demonstrated that all primers worked well, suggesting that the hedgehogs tested in this study were truly negative to Rickettsia species. We do not have Ca. Neoehrlichia DNA and did not test the Ca. Neoehrlichia primers with positive control in our laboratory although the primers has been repeatedly demonstrated to work well by other investigators [20] . Therefore, we do not know the hedgehogs were truly negative or not to Ca. Neoehrlichia. This need to be further investigated by calibrating the primers and increasing sample size. Anaplasma bovis cause disease in livestock affecting animal health and economy. The major clinical symptoms of A. bovis infection include fever, anemia, drowsiness, convulsions, weight loss, and enlargement of lymph nodes [21][22][23]. There are several reports of ruminants, raccoons, cats and deer infected with A. bovis worldwide [24][25][26][27], but information about the epidemiology of this agent is scarce in small mammals. Previous phylogenetic analysis based on groEL gene revealed that all currently available A.bovis sequences in GenBank database were divided into four lineages, and all sequences reported in China were classi ed into three lineages, suggesting a great diversity of A. bovis in and outside China [28] .
Our study revealed that A. bovis infection is common in hedgehogs and the infected individuals may be subclinical since all the molecularly detected agents were found in apparently healthy animals. The histopathological examination of the infected road-killed hedgehog also supported this inference. The maintenance of Ehrlichia involve complex zoonotic systems including ticks and reservoir hosts. Previous studies indicated wide distribution and genetic diversity of Ehrlichia in Hubei [9,[29][30][31]. However, the natural system of Ehrlichia in this region is still little known. In traditional Chinese medicine, the skin of hedgehogs has long been used in treatments for hemorrhoids, thus this species is frequently taken from the wild and eaten across central China. Also, hedgehogs frequently forage long distances and hibernate near areas where humans and domestic animals live. Thus, hedgehogs may spread ticks and tick-borne diseases to humans and animals around and in long distance.

Conclusions
We rst time detected A.bovis and a tentative new Ehrlichia species in hedgehogs collected from central China, suggesting that hedgehogs may be important in the ecology of these tick borne intracellular bacteria.

Hedgehog samples
From April, 2018 to June, 2019, hedgehogs were captured using traps baited with meat, and road killed or injured individuals were also permitted to be collected from Wuhan and Xianning cities, Hubei Province and Jiujiang city, Jiangxi Province of China (Fig.6). The hedgehogs were morphologically identi ed as Erinaceus amurensis as described previously [32]. Animals were classi ed into two age groups: young and adults. Age was estimated from the appearance of the animal, following the criteria set out by Robinson [33], gender was identi ed and recorded. Captured hedgehogs were anesthetized by an intramuscular injection (20 mg/kg) of Ketamine. One of the front feet was sterilized immediately after sedation, and one nail was clipped 3 mm short. Blood samples were immediately collected with 5ml centrifuge tubes and disposable plastic transfer pipettes. The sample volume ranged from 0.2-1ml. All blood samples were preserved with dry ice, and later stored in -80℃ for further use. All sampled individuals were later released back to the wild.

PCR ampli cation of Rickettsia, Anaplasma, Ca.Neoehrlichia and Ehrlichia
Hedgehog blood DNA was extracted with the Qiagen DNA Kit (Qiagen, Hilden, Germany). DNA concentration and purity were measured with an absorbance ratio of 260 to 280 nm by using DeNovix DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA) and were stored at -20°C until used. Mean quantity of DNA obtained was 38 ± 9.7 ng/ul and the 260/280nm ratio of all samples were 1.66 ± 0.27. Blood DNA samples were used as templates for PCR ampli cation of Ehrlichia, Ca.Neoehrlichia, Rickettsia and Anaplasma DNA with primers described in Table 1. Anaplasma 16S rRNA (rrs) gene was ampli ed with a nested PCR by using the primers EHR1/EHR2 in the rst-round reaction, and EHR3/EHR4 in the second-round reaction. The PCR conditions were set as following: denaturation for 3 min at 94℃, followed by 35 cycles of denaturation for 1 min at 94℃, annealing for 1 min at the temperatures listed in Table 1, and elongation for ~ 1.5 min at 72℃ [34]. For rrs positive samples, groEL gene was targeted for further genotyping as described previously [28]. For Ehrlichia, nested PCR ampli cations of rrs, heat sock protein gene (groEL) and citrate synthase gene (gltA) were performed [29,35]. For detection of Rickettsia, nested PCR ampli cations of Rickettsiarrs, gltA, and outer membrane protein B gene (ompB) were performed [36]. For Ca.Neoehrlichia detection, 16S rRNA were targeted [20] . DNA isolated from A.bovis, E. chaffeensis and R. japonica was served as positive controls. To avoid contamination, all steps were performed in separate rooms. Negative control with distilled water was run for each reaction. PCR products were separated with 1.2% agarose gel electrophoresis and visualized with UV light after ethidium bromide staining. PCR products with expected sizes were excised from gels and extracted using a Gel Extraction Kit (Promega, Madison, WI, USA), which were then cloned into the pMD19-T vector (TaKaRa, Shiga, Japan). M13F-47,M13R-48 Universal Primers were used for Sanger dideoxy sequencing in TingKe Biotech Company (Wuhan, China) on both strands.

Phylogenetic analysis
All sequences were searched using BLAST in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). After alignment by ClustalW with MEGA 7.0 [37], the datasets were analyzed by jModeltest2 and the best evolutionary models were chosen [38]. Phylogenetic trees were constructed using the Maximum Likelihood method with the best model in MEGA 7.0, and the robustness of the trees was tested with 1,000 bootstrap replications.

Declarations
Ethics approval and consent to participate

Competing interests
The authors declare that they have no competing interests.

Funding
This study was nancially supported by grants from National Natural Science Funds of China (Nos. 31570167). The funder had no role in study design, data collection and analysis, interpretation of data or writing of the manuscript.      The physical examination of the road-killed hedgehog. a. Ticks bite on the skin; b. Normal body appearance; c. Lung; d. Unknown worms parasitized in liver.