Molecular Detection of Rickettsia felis in Haemaphysalis longicornis Ticks in Anhui Province of China

Background Rickettsia felis causes febrile illness in humans. There is no information on the organism in Anhui Province of China. Methods Questing ticks were collected from Anhui Province of China and tick DNA was used as template for nested PCR (or real-time PCR) to amplify Rickettsia felis with 16S rRNA gene ( rrs ), 17kDa protein gene and orfB gene. Results PCR results indicated that 0.22% (2/907) ticks were positive to R. felis . Sequence analysis of the partial rrs and 17 kDa protein genes showed R. felis in ticks was highly homologous with R.felis URRWXCal2 with similarity of 98.91% and 99%, respectively. Our study demonstrated the first evidence of R. felis in H. longicornis ticks from Anhui Province of China, suggesting that a potential transmission of R. felis to humans through tick bite in China.


Introduction
Rickettsia felis, a Gram-negative obligate intracellular bacterium, is a human pathogen responsible for flea-borne spotted fever (FBSF), also known as cat flea typhus (CFT). It can infect arthropods (ticks, mites and mosquitoes) and mammals (humans, apes, gorillas, monkeys, rats, dogs, and cats) [1], and cause an acute febrile illness commonly involving headache, malaise, myalgia, rash, eschar and QIAamp® DNA Blood Mini Kit (QIAgen, Germany) following the manufacturer's instruction. The DNA samples were amplified by nested PCR with PCR primers derived from rickettsial genes including 16S rRNA (rrs), 17 kDa protein genes. PCR primers were described previously and summarized in table 1. The PCR conditions for the two genes were the same as follows: 5 min at 95℃, followed by 40 cycles of 45 s at 94℃, 45 s at 50℃, 1 min at 72℃, and a final extension at 72℃ for 10 min.
The nested PCR was conducted using 1 μL first-round PCR product as template under the following conditions: 5 min at 95℃, followed by 40 cycles of 45 s at 94℃, 45 s at 53℃, 1 min at 72℃, and a final extension at 72℃ for 10 min. The PCR products were electrophoresed in a 1.2% agarose gel and detected under UV light.
Positive amplicons were purified using agarose gel DNA extraction kit (TaKaRa, Dalian, China). The purified PCR fragments were ligated into the pMD-18T plasmid by T-A cloning, and then transformed into competent E.coli DH5α, cultivated at 37℃ for filtration of blue and white clone. Single clones were sequenced using the Sanger sequencing technique (Sangon Biotech, Shanghai, China) after colony PCR identification on both strands. All sequences were compared with the known sequence in GenBank with BLAST program.
To confirm positive results, real-time PCR targeting the orfB gene was performed using R.felis-specific primers [12] (table 1).

Phylogenetic analysis:
The DNA sequences of rrs and 17 kDa protein genes of the Rickettsia species were aligned and compared by MEGA software (Version 7). Phylogenetic trees were constructed using the Neighbor-joining method in MEGA software with 1000 bootstrap replications. A dendrogram was obtained based on genetic distances. All reference sequences were downloaded from NCBI.

Results
We collected 907 ticks from vegetation and goats, including 190 nymphs and 717 adult ticks, in five counties and one district of Anhui Province (Fig.1). All the ticks were identified as Haemaphysalis longicornis based on the morphological characters, implying H. longicornis is a predominated tick species in Anhui Province of China. The prevalence (minimum infection rate) of R. felis in each stage of the tick was determined by the assumption that a positive pool of ticks contained one R.
felis-infected tick. The prevalence of R. felis was 0 among 190 nymphs and 0.28% (2/717) among adult ticks. To further confirmation, we amplified the 17 kDa protein and orfB genes of R. felis and the results were the same as the rrs gene PCR.

Discussion
R. felis has been traditionally grouped as a member of the spotted fever group (SFG) rickettsia [15]. It was first described in the cat flea, Ctenocephalides felis, in the United States [16]. Since then, the pathogen has been detected in mosquitoes, fleas, ticks, mites and lice [3,10,17,18]. To date, thirty-nine species of arthropods have been associated with R. felis [3]. Ticks is considered one of the most important vectors of R. felis. Tick plays a key role in the epidemiology of zoonotic diseases, such as human granulocytic anaplasmosis [19]. Previous studies showed that R. felis was found to infect H. longicornis in regions of Jiangsu, China, and to infect Leptotrombidium scutellare mites in Huangdao District of Qingdao City, China [20].
The phenomenon may be attributed to the different vectors of R. felis in different ecological environments.
Anhui province located in the eastern part of China, it's an offshore inland province between east longitude 114°54 '~ 119° and northern latitude 37°41 '29 ~ 34°38' roughly, and it can be roughly divided into five regions: Huaibei Plain, Jianghuai Hill, Western Dabie Mountainous Area, Wannan Mountainous Area and the Plain along the river. In this study, we collected all the H. longicornis ticks from the mountainous areas owing to almost no ticks in the plain areas. We detected R. felis from these ticks with nested PCR (or real-time PCR) based on 16S rRNA, 17kDa and orfB genes.
As we all known, sequence comparison of the 16S rRNA gene is considered one of the most powerful and precise molecular methods for analyzing the phylogenetic relationships of bacteria. In this study, the 16S rRNA gene sequences of the positive tick samples from Anhui Province were closely related to R. felis (Fig.1).
Additionally, differences in sequencing results indicate that there may be a few of genetic variants in Anhui Province, even simultaneously in the same region. As can be seen from Figure 1, the two 16S RNA sequences from Anhui Province were all identical to R. felis URRWXCal2 (CP000053.1). However, phylogenetic analysis of 17kDa protein gene indicated that R. felis strain Anhui-1 detected in ticks from Anhui Province, was more similar with R.felis isolates (MF491767.1 and AF195118.1), while R. felis strain Anhui-2 was more similar with these isolates (MF491774.1, MF491775.1 and MF491778.1), implying R. felis in China is genetically diverse (Fig. 2). For further confirmation, qPCR was carried out using specific R. felis primers targeting orfB gene to detect the positive samples. Through sequence analysis, all the positive samples were R. felis, and the nucleotide sequences of orfB from the ticks were identical to each other.  Figure 2