Detection of Rickettsia raoultii in Vermipsylla alakurt-Like Fleas of Sheep in Northwestern China

Acta Parasitologica (2024) 69:776–784 https://doi.org/10.1007/s11686-024-00809-y ORIGINAL PAPER Detection of Rickettsia raoultii in Vermipsylla alakurt‑Like Fleas of Sheep in Northwestern China Fengshi Li1 · Shanshan Zhao1 · Ente Li1 · Songsong Xie2 · Nan Wang1 · Wenbo Tan1 · Yuanzhi Wang1 Received: 21 May 2023 / Accepted: 19 January 2024 / Published online: 28 February 2024 © The Author(s) 2024 Abstract Introduction To date, a total of 2574 validated flea species have been discovered. Vermipsyllidae is a family of fleas that comprises at least eight species. Vermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Here a novel Vermipsylla species was described, and rickettsial agent was also detected in it. Methods A total of 128 fleas were collected directly from 260 pastured sheep in China. Of these, eight representative fleas (four males and four females) were identified by key morphological features. Meanwhile, 120 flea DNAs, including six flea samples for molecular taxonomy, were subjected to Rickettsia spp. DNA detection. The molecular identity of fleas was determined by amplification and sequenmce analysis of four genetic markers (the 28S rDNA genes, the 18S rDNA genes, the mitochondrial cytochrome c oxidase subunit I and subunit II). In addition, five Rickettsia-specific gene fragments were used to identify the species of the rickettsial agents. The amplified products were sequenced and phylogenetically analyzed. Results The morphological characteristics of the flea species identified in this study were similar to Vermipsylla alakurt, but presented difference in hair number of the metepimeron, the third tergum, the genitals and the tibiae of hind leg. The 18S rDNA, 28S rDNA and COII genetic markers from fleas showed the highest identity to those of V. alakurt, shared 98.45% (954/969), 95.81% (892/931) and 85.86% (571/665) similarities, respectively. However, the COI sequence showed the highest identity to that of Dorcadia ioffi with 88.48% (576/651) similarity. Rickettsia raoutii tested positive in 14.17% (17/120) flea DNA samples. Conclusion Our study reports the detection of R. raoultii in V. alakurt-like fleas infesting sheep in China. Keywords Rickettsia raoutii · PCR · Vermipsyllidae · Fleas · Sheep · Northwestern China Introduction Fengshi Li, Shanshan Zhao and Ente Li contributed equally to this work. * Wenbo Tan * Yuanzhi Wang 1 Key Laboratory for Prevention and Control of Emerging Infectious Diseases and Public Health Security, the XPCC, School of Medicine, Shihezi University, Shihezi City 832002, Xinjiang Uygur Autonomous Region, People’s Republic of China 2 The First Affiliated Hospital of Shihezi University Medical School, Shihezi City 832002, Xinjiang Uygur Autonomous Region, People’s Republic of China Vol:.(1234567890) Fleas (Insecta: Siphonaptera) are small, laterally flattened, wingless, and highly specialised insects [1], which belong to arthropod phylum, insecta, Siphonaptera. Currently, at least 2575 validated flea species belonging to 16 families and 246 genera have been described [2]. Vermipsylla, a genus of the family Vermipsyllidae, includes eight validated species, i.e. Vermipsylla alakurt (Kazakhstan, Mongolia, China), V. asymmetrica (China), V. ibexa (China), V. minuta (China), V. parallela (China), V. perplexa (China, Nepal), V. quilianensis (China) and V. yeae (China) [3, 4]. V. alakurt was firstly identified in China in 1965, in the southern region of Xinjiang Uygur Autonomous Region (XUAR), northwestern China [5]. During December to March, the adult fleas are mainly endemic in alpine pastoral areas, and prevailingly infest sheep, yaks, horses, yellow cattle and some Acta Parasitologica (2024) 69:776–784 wildlife species, causing irritation, poor condition, anaemia, abortion and even death [6–8]. Fleas are of tremendous medical and economic importance as vectors of several diseases important to human health including bubonic plague, murine typhus, and epidemic typhus [9, 10]. Rickettsia typhi in Ctenocephalides felis, Rickettsia felis in Liposcelis bostrychophila and Candidatus Rickettsia barbariae in V. alakurt, were previously reported [11–13]. Materials and Methods Sample Collection and Identification of Fleas In January 2018, fleas (128 in total) were collected directly from the entire body of 260 pastured sheep from two sheep flocks in Altaw Mountain, Wenquan County (the north region of XUAR, 2200 m a.s.l; 44°470ʹ30 N, 80°53ʹ30 E), which was adjacent to Kazakhstan [14]. The collected fleas were divided into two parts. Eight representative fleas (four males and four females) were for morphological identification by Stereomicroscope according to key features [13, 15] (eg. body length, labial palpus and notch of the tibiae of hind leg). In addition, DNAs of the individual fleas were extracted using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Six DNA from six representative flea samples were subjected to PCR amplification of four genetic markers [the 28S rDNA gene, the 18S rDNA gene and the mitochondrial cytochrome c oxidase subunit I (COI) and subunit II (COII)] for molecular identification. The nucleotide sequence were manually edited and compared to GenBank reference sequences (http://www.ncbi.nlm.nih.gov/ BLAST/). Phylogenetic trees were constructed by the MEGA 7.0 software with the Maximum Likelihood (ML) method [16]. Detection of Rickettsial Agents and Sequence Analysis A total of 120 flea DNAs, including six flea samples for molecular taxonomy, were subjected to PCR amplification for the detection of Rickettsia spp. DNA. Five rickettsial genetic markers, 17-kilodalton antigen 17-kilodalton antigen (17-kDa), surface cell antigen 4 (sca4), citrate synthetase (gltA), surface cell antigen 1 (sca1), and outer membrane proteins A (ompA) were used according to published protocols [17, 18]. Each PCR assay included a negative control (distilled water instead of flea DNA template) and a positive control (DNA from Candidatus R. barbariae obtained from V. alakurt). The above procedures were applied to treat the PCR products and their corresponding sequences. 777 A phylogenetic tree was constructed by the MEGA 7.0 software with the ML method [13]. Results A total of 128 fleas were collected from the entire body of 260 sheep (10 males, 250 females) in two flocks in Altaw Mountain, Wenquan County, which is adjacent to Kazakhstan [14]. The fleas were divided into two parts: eight fleas (four males and four females) were preserved for morphological identification, and the remains were used for other purposes. The collected fleas had the following morphological characteristics similar to V. alakurt, which can be clearly distinguished from the other seven Vermipsylla species (shown in Table 1). Its size was the largest in the members of Vermipsylla genus, with males measuring 3.7–4.9 mm and females measuring 5.6–7.5 mm or longer. It (...truncated)