Molecular detection of spotted fever group rickettsiae in ticks parasitizing pet dogs in Shihezi City, northwestern China

Experimental and Applied Acarology (2019) 77:73–81 https://doi.org/10.1007/s10493-018-00337-1 Molecular detection of spotted fever group rickettsiae in ticks parasitizing pet dogs in Shihezi City, northwestern China Wurelihazi Hazihan1 · Zhihui Dong2 · Liping Guo3 · Kadyken Rizabek4 · Dzhunysov Askar4 · Kulmanova Gulzhan4 · Mahanov Kudaibergen4 · Akishev Nurlan Kenjebaevich4 · Tolegen Talgat4 · Kenesbay Kairullayev4 · Yuanzhi Wang2 Received: 18 July 2018 / Accepted: 28 December 2018 / Published online: 16 January 2019 © The Author(s) 2019 Abstract A total of 178 adult ticks were collected from 32 pet dogs from five veterinary clinics in Shihezi City, Xinjiang Uygur Autonomous Region (XUAR), northwestern China. All the ticks were identified by comprehensive morphological and genetic analyses, and rickettsiae were detected by seven Rickettsia-specific genetic markers in the ticks. The ticks collected were identified as Rhipicephalus sanguineus sensu lato. Twenty-one of the 178 samples (11.8%) were positive for rickettsiae. Among these, in 13 (61.9%) samples Candidatus R. barbariae were identified, in five (23.8%) samples R. massiliae, and in three (14.3%) samples R. conorii. This study indicates that more attention should be paid to rickettsial infection in pet dogs and their ticks, because the latter may pose an epidemiological risk for tick-borne transmission of rickettsiae to human beings. Keywords Rhipicephalus sanguineus sensu lato · Spotted fever group rickettsiae · Pet dogs · Northwestern China Introduction Ticks are among the most common ectoparasites of dogs, also involved in the transmission of a number of major diseases in both dogs and humans (Chomel 2011; Dantas-Torres and Otranto 2016). Tick-borne rickettsioses are caused by the spotted fever group rickettsiae (SFGR) of the genus Rickettsia, which contains approximately 20 species, and many of which are established or emerging human pathogens (Wood et al. 2012). Besides, more and Wurelihazi Hazihan, Zhihui Dong and Liping Guo are equal contributors. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1049 3-018-00337-1) contains supplementary material, which is available to authorized users. * Yuanzhi Wang Extended author information available on the last page of the article 13 Vol.:(0123456789) 74 Experimental and Applied Acarology (2019) 77:73–81 more new SFGR species have been found across the world, as a result of range expansion of tick populations, changes in landscape and climate, and more accurate diagnostic testing (Trotta et al. 2012; Yunik et al. 2015). Due to the emerging and re-emerging nature of tick-borne diseases in humans, increasing focus has been placed on research of ticks parasitizing domestic animals (Hiraoka et al. 2005). As in many other countries, in China the dog has become a bonded family member. Regardless the benefits of having pet dogs, pathogens carried by ticks are potentially transmissible to humans, which may represent a health risk, especially to children, elderly people and immunocompromised individuals (Dantas-Torres and Otranto 2014). To date, at least three protozoan (Theileria, Babesia and Hepatozoon) and five bacterial (Anaplasma, Ehrlichia, Rickettsia, Coxiella and Bartonella) tick-borne genera have been reported in domestic dogs around the globe (Beck et al. 2009; Brown et al. 2006; Buhariwalla et al. 1996; Camacho et al. 2001; Conrad et al. 1991; Kaewkong et al. 2014; Kamani et al. 2013; Levin et al.2012; Mokhtar et al. 2013; Yabsley et al. 2008). In Jiangxi Province, mid-eastern China, Babesia canis vogeli and Babesia gibsoni were molecularly detected in 780 dog ticks (749 Rhipicephalus sanguineus, 16 Haemaphysalis campaulata and 15 Haemaphysalis verticalis), while all sampled dog ticks were negative for rickettsial agents (Zheng et al. 2017). In Xinjiang Uygur Autonomous Region (XUAR), northwestern China, rickettsial agents were prevalent in ticks infesting both domestic animals and wildlife (Guo et al. 2015, 2016). However, there is limited knowledge on the species of ticks infesting dogs. Here a molecular investigation was carried out for rickettsial agents in pet dog ticks. Materials and methods Collection and identification of ticks In 2016–2017, ticks were sampled from 32 pet dogs presented at five veterinary clinics with symptoms of depression, weight loss and anorexia in Shihezi City (483 m above sea level, at 44°268129ʹN 86°0627148ʹE), the northwestern China. The ticks were placed in tubes with 75% ethanol and stored at − 80 °C. All of the ticks were identified morphologically according to previous reports (Filippova 1997; Dantas-Torres et al. 2013a, b). Twenty-nine representative ticks, with 4–6 ticks at each veterinary clinic, were used to analyze tick species and genetic diversity based on partial mitochondrial 16S rRNA (460 bp), 12S rRNA (400 bp) and coxI (889 bp) gene sequences (Szabó et al. 2005; Chen et al. 2014). DNA extraction and molecular detection After detailed morphological analysis, genomic DNA was extracted from each individual tick using the TIANamp Genomic DNA Kit (TianGen, Beijing, China). The ticks were mechanically crushed twice in sterile water for 15 min and then dried on sterile paper, suspended in 200 µl tissue lysis buffer and 40 µl proteinase K (100 µg/ml), and incubated overnight at 56 °C. The final elution volume was 60 µl. Subsequently, the polymerase chain reaction (PCR) technology was used to detect rickettsial agents with seven genetic markers for DNA fragments [434-, 1332-, 1060-, 488-, 920-, 491-, and 812-bp products of the genes encoding the 17 kilodalton antigen (17-kDa), 16S rRNA(rrs), citrate synthase (gltA), surface cell antigen 1 (sca1), PS120-protein-encoding gene (gene D), and outer membrane proteins A and B (ompA and ompB)] (Anstead and Chilton 2013; Chilton 2013; Sekeyova 13 Experimental and Applied Acarology (2019) 77:73–81 75 et al. 2001; Wei et al. 2015). (Table 1). Rickettsia aeschlimannii from Rh. turanicus and double-distilled water were used, respectively, as positive and negative controls (Wei et al. 2015). The PCR products were purified using the TIANgel Midi Purification Kit (TIANGEN, Beijing, China), and then subjected to sequencing (BGI, Shenzhen, China). Phylogenetic analyses were conducted used MEGA version 6.0 based on the 17 kDa-rrsgltA-ompA-ompB-gene D concatenated sequence data of the rickettsiae by Maximum Likelihood (ML) and Neighbor-Joining (NJ) methods (Tamura et al. 2013). Results A total of 178 adult ticks (76 males and 102 females) were collected and morphologically identified as Rh. sanguineus sensu lato. (Fig. 1). The sequencing data from the 29 representative ticks confirmed the morphological results based on Basic Local Alignment Search Tool (BLAST) analyses of 16S rRNA, 12S rRNA and cox1. Rhipicephalus sanguineus s.l. in this study had 93.3–93.8% pairwise nucleotide sequence identity to genome sequences of the reference (...truncated)