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

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.


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.
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 3.7-4.9mm and females measuring 5.6-7.5 mm or longer.Its labial palpus was no longer than 17 segments, with 12-15 segments in females and 10-14 segments in males.The rear part of the female flea was full of fat, making up about three-quarters of its body length.The head of the intromittent organ of the males looked like a winter glove (with the back four fingers held together).The head of the spermathecae is ellipsoid, and the tail part was thin and long, with a sausage-like shape.The above morphological characteristics was similar to V. alakurt, interestingly, the females had less hairs (n = 5) next to the notch of the tibiae of hind leg, which obviously distinguishes from V. alakurt (n = 13).The differences from other key morphological features, eg. the hair of the metepimeron, the hair of the third tergum, the hair of the tibiae of hind leg and the hair of the genitals, were shown in Fig. 1 and Table 2. Due to the limited data available in GenBank, only two Vermipsyllidae species, V. alakurt and Dorcadia ioffi, were compared.The sequenced PCR product of 18S rDNA, 28S rDNA and COII obtained from fleas in this study were 98.45% (954/969), 95.81% (892/931) and 85.86% (571/665) similarities to V. alakurt (GenBank accession number KR297206, KR297207, KT193612), respectively.However, the COI nucleotide sequence of fleas in this study were 91.16% identical to D. ioffi in GenBank (accession number MF124314).Based on morphological and molecular evidence, all the fleas were identified as a novel Vermipsylla species, here named as Vermipsylla alakurt-like.

Discussion
Combined with molecular identity, systematic morphological identification in arthropods is important for the identification of a new species or subspecies [19,20].To date, morphology can also be used as a helpful tool to distinguish flea species or subspecies, such as C. felis and C. canis [21].The flea species identified in this study can be classified into the Vermipsylla genus according to some morphological key features, such as no combs, its labial palpus less than 17 segments, the tibiae of hind leg having 6 notches, full of fat at the rear 3/4 part of the female body, only one seminal vesicle and its head being ellipsoid [22].Although COI of Vermipsylla alakurt-like shared higher similarity (88.48%) with D. ioffi than datum between Vermipsylla alakurt-like and V. alakurt, we still believed Vermipsylla alakurt-like presents its own characteristics.Interestingly, Vermipsylla alakurt-like and V. alakurt have some similarities, such as segment number (n = 10-15) of labial palpus, the aedoeagus (in the shape of a winter glove) and the spermathecae (the head is ellipsoidal, and the tail is slender and sausage-shaped).Meanwhile, there are also some differences, especially in the number of hair and bristle of metepimeron, the third tergum, the tibiae of hind leg, aedoeagus and spermathecae (shown in Table 2), which makes it distinct from the other eight validated species of Vermipsylla genus, including V. alakurt.This finding indicates that the morphological characteristics (eg. the hair number of the metepimeron, the third tergum, the genitals and the tibiae of hind leg) could be helpful in identification and taxonomy especially for Vermipsylla species.
Herein, we reported the presence of R. raoultii in Vermipsylla alakurt-like fleas from sheep in an alpine pastoral area in the north-western of China.There is not enough evidence V. alakurt-like, male.6b: the whole body of V. alakurt, male.7a: the metepimeron of V. alakurt-like, male.7b: the metepimeron of V. alakurt, male.8a: the third and fourth tergum of V. alakurt-like, male.8b: the third and fourth tergum of V. alakurt, male.9a: the tibiae leg of V. alakurt-like, male.9b: the tibiae leg of V. alakurt, male.10a: the aedoeagus of V. alakurt-like, male.10b: the aedoeagus of V. alakurt, male to confirm that Vermipsylla alakurt-like can transmit rickettsiosis to sheep.In future, detecting R. raoultii in sheep organs would demonstrate its potential to cause the disease.Previously, the special geographical environment and current research confirms that R. raoultii is highly prevalent in the northwest of China and its neighboring countries, such as Mongolia, Russia and Kazakhstan [22,42,43].We should pay attention to the transmission of SFGR in a wider arthropod species especially in the blood-suckers.

Conclusion
A novel Vermipsylla species from alpine pastoral region was described.This is the first report of the presence of R. raoultii in Vermipsyllidae.These findings extend our knowledge of Vermipsylla species, and the geographical distribution and reservoir hosts for R. raoultii.Future research should assess the vector competence and Table 2 The difference in the amount of hair and bristle among V. alakurt (male), V. alakurt-like (male), V. alakurt (female) and V. alakurt-like (female) in metepimeron, the third tergum, the fourth tergum, tibiae leg, aedoeagus and spermathecae