Seroepidemiological Study of Spotted Fever Group Rickettsiae and Identification of a Putative New Species, Rickesttsia sp. Da-1, in Gongliao, Northeast Taiwan

Tick-borne spotted fever group (SFG) rickettsioses were neglected in Taiwan. The study reported a seroepidemiological survey of SFG rickettsiae in residents in Gongliao District, Northeast Taiwan. Blood samples were examined for antibodies against SFG rickettsiae by enzyme-linked immunosorbent assay and immunofluorescence assay. Risk factors were assessed using logistic regression. Ticks parasitizing dogs were collected within a 2 km radius from the houses of seropositive participants, and PCR was performed to detect possible tick-borne pathogens. Of 1108 participants, 75 (6.8%) had antibodies against SFG rickettsiae. Residents were more likely to be seropositive if they were older than 65 years, recruited by Dr. Enjoy’s Clinic, or resided in Jilin village. A total of 184 ticks including 5 species (Rhipicephalus sanguineus, Rhipicephalus haemaphysaloides, Dermacentor auratus, Haemaphysalis hystricis, Haemaphysalis ornithophila) were collected. Rickettsia spp. were detected in 6.5% (12/184) of ticks. Rickettsia sp. TwKM01 was found in 6 R. sanguineus and 4 R. haemaphysaloides; while Rickettsia sp. TwKM03 was identified in 1 R. sanguineus. Moreover, gene-based pairwise analysis indicated identification of a putative new species, Rickettsia sp. Da-1, in D. auratus. These findings provided evidence of SFG rickettsiae infection in ticks and suggested SFG rickettsiae exposure in the residents.


Introduction
Rickettsiae are obligate intracellular Gram-negative bacteria belonging to the order Rickettsiales [1]. Genus Rickettsia was classified into 3 groups: spotted fever group (SFG) rickettsiae, typhus group (TG) rickettsiae, and scrub typhus group based on serology [2]. Later, the scrub typhus group was designated as a new genus, Orientia [3]. Further genetic evidence has suggested the addition of a transitional group (TRG) along with the ancestral group (AG) rickettsiae [4]. SFG rickettsiae and AG rickettsiae are primarily transmitted by ticks; while TG rickettsiae and TRG rickettsiae are associated with fleas, chiggers, or lice [5].
In Taiwan, the notifiable rickettsial diseases cover scrub typhus, epidemic typhus, and murine typhus. Scrub typhus and murine typhus led to 300-500 and 20-60 confirmed cases each year, respectively; while epidemic typhus has not been documented since World War II [6]. The fact that only 13.1-19.9% of the blood samples sent to the Taiwan CDC for laboratory diagnosis of scrub typhus were actually positive for Orientia tsutsugamushi enrolled by the group practice center inhabited the northeastern Gongliao and the coastal villages; while subjects enrolled by Dr. Enjoy's Clinic mostly lived in the southwestern Gongliao (Table 1).

Potential Risk Factors for SFG Rickettsiae Exposure
Univariable logistic regression revealed significantly positive association between SFG rickettsiae IgG seropositivity and older age, patients visiting either the group practice center or Dr. Enjoy's Clinic versus healthy individuals, living in Jilin village, and the job of industrial laborer (Table 3). In multivariable logistic regression, seropositivity remained significantly associated with age (p = 0.014). Sera positive for IgG against SFG rickettsiae were 2.1 times more likely to be collected from participants ≥ 65 yearsold (95% CI = 1.2-3.8) and 3.3 times more likely to be from inhabitants of Jilin village. Reactivity to multiple groups of rickettsiae was also positively associated with older age (p = 0.009) and patients of the group practice center (95% CI = 1.2-53.3) or Dr. Enjoy's Clinic (95% CI = 2.8-255.6).

Collection of Ticks
To further understand the transmission of SFG rickettsiae in the area, ticks were collected from dogs living within 2 kilometer radius from the participants having IgG against SFG rickettsiae. Of 72 dogs encountered, ticks were found on 14 dogs, giving an infestation rate of 19

Discussion
Here we report an extensive study of Rickettsia exposure in the northeastern part of Taiwan. The community-based seroepidemiological survey including 1108 residents in Gongliao District revealed seroprevalence of 6.8% (75/1108) for SFG rickettsiae, 4.1% (45/1108) for TG rickettsiae, and 14.0% (115/1108) for O. tsutsugamushi. Ticks were collected from dogs living near the seropositive participants to look for potential tick-borne pathogens. Rickettsia spp. were detected in 6.5% (12/184) of the ticks. One putative new species similar to R. raoultii and "Candidatus R. laoensis" was discovered in D. auratus according to the criteria for molecular identification of Rickettsia [37]. Whether these microorganisms lead to human diseases remains to be investigated.
Our study showed 20.0% (222/1108) of the serum samples collected from residents in Gongliao District reacted to at least one group of rickettsiae. Considering IFA of R. rickettsii as the gold standard, the sensitivity and specificity of SFG ELISA were 97.4% and 95.8%, respectively. Forty-three of 222 (19.4%) reactive samples were positive to more than one group of rickettsiae. Cross-reactivity has been known to occur between SFG rickettsiae and TG rickettsiae, especially IgG antibodies [38]. Titration of sera was supposed to be performed to reveal differences in antibody titers for the distinction between crossreactivity [39]. However, the observed IgG titers were generally quite low (≤256), making comparison using titration unrealistic. Therefore, we were unable to determine whether the reactivity was caused by exposure to different groups of rickettsiae or the result of cross-reactions. Moreover, sera from patients with R. felis infection were shown to react to R. rickettsii and R. conorii, and R. felis has been proposed to be the major cause of crossreactions between R. typhi and R. conorii [40,41]. A study detected R. felis infection in 21.6% of patients with rickettsioses in North Taiwan [12]. Hence, R. felis exposure was speculated to be responsible for some of the reactions although the current study did not include R. felis in the assay. Conversely, cross-reactions between SFG rickettsiae/TG rickettsiae and scrub typhus were less common. Only 5 samples reacted to both R. rickettsii and O. tsutsugamushi, and 5 samples reacted to both R. typhi and O. tsutsugamushi. The prevalence of IgG antibodies against SFG rickettsiae, TG rickettsiae, and multiple groups of rickettsiae was significantly associated with older age, implicating progressive exposure to Rickettsia spp. Samples collected from Dr. Enjoy's Clinic tended to have higher seropositive rates probably due to the older average age of the patients. Moreover, a part of the patients from Dr. Enjoy's Clinic was receiving home-based medical care voluntarily provided by the clinic. These patients generally lived in remote areas and belonged to a disadvantaged minority. As shown in the risk analysis, inhabiting the southwest village, Jilin, was a risk factor for seropositivity.
Although some scenic spots in Gongliao District have become popular attractions for tourists, most parts of the district remained rural. Dogs shuttling back and forth between the fields and human houses, making them a bridge to wildlife as well as their ectoparasitic ticks. In this case, the dogs were considered as sentinels and ticks were collected from the dogs living near seropositive participants. Most collected ticks were the brown dog ticks (R. sanguineus) and R. haemaphysaloides (155 and 24, respectively), which was generally consistent with other study [42], but species such as D. auratus, H. ornithophila, and H. hystricis also appeared in the collection. Rickettsia sp. TwKM01 and Rickettsia sp. TwKM03 were detected in R. sanguineus and R. haemaphysaloides. Phylogenetic analyses revealed Rickettsia sp. TwKM01 was most similar to Rickettsia rhipicephali [21]. Rickettsia sp. TwKM03, a species close to R. felis, was first identified in Leptotrombidium chigger mites and widely distributed in I. granulatus in Hualien, Kinmen, and Matsu in Taiwan [21]. The species has also been found to infect R. sanguineus and Ctenocephalides felis collected from dogs in Brazil [43]. Moreover, the sequencing results indicated a putative new species, Rickettsia sp. Da-1, was identified in D. auratus. Pairwise comparison revealed the amplified fragments of gltA and sca4 were 98.4% and 98.8% identical to "Candidatus R. laoensis", respectively, which were lower than the gene sequence-based criteria of identification (99.9% and 99.3%, respectively) despite the 100% similarity of sequences from 5' end of ompA [37]. The amplicons of ompB were highly similar to "Candidatus R. laoensis", but the reference sequence was only 1109 base pairs. "Candidatus R. laoensis" was first discovered in a pool of Haemaphysalis ticks in Laos [36]. Later the Rickettsia was observed in 63.6% (14/22 pools) of ticks (Haemaphysalis bispinosa, H. flava, H. hystricis, Haemaphysalis longicornis, Dermacentor atrosignatus, D. auratus, D. taiwanensis, Dermacentor silvarum) infesting wild boars in Southeast China. However, the study used only partial ompA for the screening for infection [44]. The tick host in our study, D. auratus, has not previously been documented in Taiwan, but our follow-up studies confirmed the continuous existence of the species on the island (unpulished data). Dermacentor auratus has been found to parasitize wild boar, cattle, deer, buffalo, and small mammals in other countries, and cases of human infestation were recorded [45,46]. According to a personal communication, a female residing next to Gongliao District attended a clinic for being attacked by D. auratus. Whether D. auratus ticks can transmit rickettsiae to humans or small mammals and maintain rickettsiae in nature remains to be studied. On the other hand, one of the limitations of the current study resulted from our method of tick collection. For example, I. granulatus, which is abundant and has been shown to carry Borrelia and Rickettsia [22,47], was left out along with many other ticks known to occur in Taiwan. However, I. granulatus mainly parasitizes small mammals and there is no record of it attacking humans in the Australasian Zoogeographic Region [48]. Considering the terrain, the animal contact history, and the less active lifestyle of the participants, ticks were collected only from dogs in the study. Further surveys of ticks using different collection methods would provide more information regarding ticks and tick-borne pathogens in the region.
Tick-borne rickettsioses have been considered as important infectious diseases in the Western World. For example, Rocky Mountain spotted fever and human granulocytic anaplasmosis are nationally notifiable diseases in the USA [49]. Thirteen EU countries have established a surveillance system at the national level and reporting of rickettsioses are mandatory [50]. In Asia, Japanese spotted fever is a notifiable infectious disease in Japan, with approximately 200-300 cases reported annually and once a seropositive rate of 45.1% in Okinawa [51]. New pathogenic species, such as Rickettsia heilongjiangensis, R. helvetica, and Rickettsia tamurae, and other species with unknown pathogenicity, such as Rickettsia asiatica and "Candidatus Rickettsia tarasevichiae", have been identified after R. japonica [52][53][54][55][56]. In South Korea, Thailand, Malaysia, and Laos, seroprevalence for SFG rickettsiae has been described as 16.2-19.9%, 0.8-4%, 42.5%, and 2.6% in patients with acute febrile illness, respectively [57][58][59][60][61][62]. The seroprevalence was reported as 1.7% and 10.4-20.4% in healthy populations in Vietnam and Indonesia, respectively [63,64]. Spotted fever is a common disease in China, and the seroprevalence has been demonstrated as high as 54.8% in the healthy population in Eastern China [65]. New species, including R. heilongjiangensis and Rickettsia sibirica mongolotimonae, have also been identified [37,66].
In Taiwan, tick-borne SFG rickettsioses are still poorly understood. Our findings confirmed the exposure of SFG rickettsiae, with a seropositive rate of 6.8% in residents of the northeastern part of the island. Rickettsia spp. and other endosymbionts were identified in ticks, implying the existence of transmission cycles. Although the pathogenicity of these rickettsiae remained uncertain, SFG rickettsiae infection should be considered in the differential diagnosis of fever of unknown origin in addition to scrub typhus. This work emphasized the risk of tick-borne rickettsioses. Residents as well as tourists should wear personal protection equipment or repellent when engaging in outdoor activities, and practicing tick control in dogs is advised. Further investigations will focus on the patients to detect and isolate SFG rickettsiae for the verification of etiological agents of febrile illnesses. Other tick-borne pathogens, such as severe fever with thrombocytopenia syndrome virus, will also be explored.

Study Setting and Human Subject
Gongliao District is a rural district located in the northeastern part of Taiwan. The district comprises an area of approximately 99.97 square kilometers which is divided into 11 villages. The population was 13,970 in 2008 with 73.6% of the inhitants dwelling in Zhenli, Renli, Hemei, Fulong, Goungliao, and Fulian villages. The terrain is generally hilly. Facing the Pacific Ocean, Gongliao District is famous for its golden-sand beach, the Fulong beach, and other scenic settings as well as old hiking trails. Tourists come to the district for seafood or recreation activities, such as surfing, windsurfing, fishing, hiking, and the music festival.
Human subjects were recruited through 3 sources, including healthy individuals attending physical examinations in a group practice center, non-febrile patients visiting the group practice center, and patients visiting or receiving home-based medical care provided by a clinic. Residents of Gongliao District, which covered workers who had been staying there for over 3 months, were invited to particpate in the study. Blood samples were collected via venipuncture after obtaining the subjects' consent and left to stand for 30 min at room temperature. Sera were separated by centrifugation, aliquoted, and carried back to the laboratory on ice. The samples were then kept frozen at −20 • C until further analyses. Meanwhile, willing participants were asked to fill out paper-based questionnaires to provide demographic information for further risk analysis. All participants did not exhibit typical signs of acute infections at the time of sampling.  [19]. Briefly, sera were diluted to 1:32 and applied to the wells. After 30 min of incubation at 37 • C, the slides were washed with PBS. Fluorescein isothiocyanate (FITC) conjugated goat antihuman IgG/A/M (ThermoFisher Scientific Inc., Camarillo, CA, USA) was then added. After incubation, the slides were washed, dried, and mounted. The results were read at a magnification of 400× with a fluorescence microscope (Leica Microsystem, Singapore) by two technicians independently. An antibody titer of ≥1:64 was considered as a positive reaction.

Serology
Antibodies against O. tsutsugamushi were screened by indirect IFA using slides coated with whole-cell antigens of the Karp strain as described [67,68]. A titer ≥ 1:64 was considered seropositive and indicated potential exposure to O. tsutsugamushi.

Collection of Ticks and Tick Species Identification
Ticks parasitizing dogs were collected within a 2 km radius from the houses of participants who tested positive for SFG rickettsiae antibodies during August to December, 2009. Ticks were removed by forceps or a tick twister after obtaining agreement from the dog owners and stored in 70% ethanol. Species identification was carried out by oberserving the taxonomic characteristics under a dissecting microscope [69][70][71][72]. Molecular identification targeting the mitochondrial 16S rDNA and COI was applied to nymphs whose morphological features were not fully developed and adults whose key characteristics were undistinguishable due to damages during removal or engorgement with blood for confimative purpose [69,73,74]. However, the coding sequences of COI were not efficiently amplified in nearly half of the specimens by PCR. Therefore, molecular identification was primarily dependent on the amplicons of the 16S rDNA. In addition, the 16S rDNA of ticks other than R. sanguineus and R. haemaphysaloides were sequenced to provide further information for the genetic charateraterization.

Detection of Potential Tick-Borne Pathogens
The collected ticks were cut symmetrically into halves, and a half of each tick was used in DNA extraction with Genomic DNA Mini Kit (Geneaid, Taipei, Taiwan). PCR was performed to detect potential pathogens carried by ticks. Identification of rickettsial infections was achieved by primers targeting ompA, ompB, gltA, and sca4 as previously described [75][76][77][78][79]. PCR products were sent to Mission Biotech (Taipei, Taiwan) for Sanger sequencing in both forward and reverse directions.

Phylogenetic Analysis
Sequences of the amplicons were first examined with Seqman 7.1.0 (Lasergene, Madison, WI, USA). Then the sequences were seperately aligned against those closely related species found in GenBank using the Clustal W application within BioEdit 7.2.0. Phylogenetic trees were constructed based on the alignment using a maximum likelihood inference with 1,000 bootstrap replicates in MEGA7 [80]. Novelty of Rickettsia sp. was determined by the sequence identities of the pairwise comparison of gltA, ompA, ompB, and sca4 sequences with their closest related species [37].

Statistical Analysis
The demographic information and serological results of participants were inputted to a Microsofft Excel (Microsoft Corporation, Washington, DC, USA) datasheet for further processing. A Kruskal-Wallis test (non-parametric test) was done to compare the geometric ratios between groups with a significance level of 0.05. Associations between subject char-acteristics and serological results were studied using univariable and multivariable logistic regression, using the odds ratios and 95% Confidence Interval (CI) as measurement. All variables were treated as categorical. Multivariable analysis included variables which were significantly associated with seropositivity in the univariable analysis. Effect modification and interaction were assessed for each covariate associated with seropositivity. Statistical analyses were performed using SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA).