First report of Anaplasma ovis in pupal and adult Melophagus ovinus (sheep ked) collected in South Xinjiang, China

Background Melophagus ovinus (sheep ked) is a blood-feeding ectoparasite that belongs to the family Hippoboscidae (Diptera: Hippoboscoidea) and mainly parasitizes sheep. The life-cycle of M. ovinus consists of three stages: larva, pupa and adult. It has a worldwide distribution and has been found in four provinces of China, especially South Xinjiang. In addition to causing direct damage to animal hosts, M. ovinus serves as a vector for disease transmission. In this study, our aim was to investigate the presence of Anaplasma spp. in pupal and adult M. ovinus. Methods A total of 93 specimens (including eight pupal specimens) of M. ovinus collected in South Xinjiang were selected for isolation of genomic DNA, followed by PCR amplification and sequencing of the msp4 gene of Anaplasma spp. The sequences were analyzed in MEGA 7.0 software and via online BLAST. Results PCR and sequencing results showed that all the specimens collected in 2013 were free of Anaplasma spp., whereas three and 25 specimens (including five pupal specimens) collected in 2016 and 2017, respectively, tested positive for Anaplasma spp. The analysis of 24 msp4 gene sequences (from four pupal specimens) confirmed the presence of A. ovis in M. ovinus specimens collected in South Xinjiang, China. The detected A. ovis isolates belong to Genotypes II and III. Conclusions To the best of our knowledge, this is the first report of the detection of A. ovis DNA in pupal M. ovinus, confirming the vertical transmission of A. ovis in M. ovinus and the potential of M. ovinus to serve as a vector for A. ovis.


Background
Melophagus ovinus (sheep ked), is a blood-feeding ectoparasite that belongs to the family Hippoboscidae (Diptera: Hippoboscoidea) and has significant economic effects [1,2]. Melophagus ovinus (Fig. 1a, b) is an approximately 4-6 mm long wingless fly with a small head, strong and sharp mouthparts, an oval or round abdomen, dense bristles on the body surface, and three pairs of legs tipped with claws [2,3].
The life-cycle of M. ovinus consists of three stages: larva, pupa (Fig. 1c) and adult [1,4]. Six to eight days after mating, the female fly produces larvae that adhere to the body surface of hosts and are ready to pupate into brown pupae within 6-12 hours. After 19-30 days, the pupae develop into adults, which parasitize the body surface of sheep [1].
Melophagus ovinus is widely distributed and has been found in many European, African, Asian, Oceanian, and North American countries [2]. Until now, M. ovinus has been reported to parasitize only sheep and Tibetan antelopes in Xinjiang [2,5], Qinghai [2,6] and Gansu [3,7] in China. Additionally, adult or pupal M. ovinus specimens have been detected on imported sheep and sheep skin and wool during port-quarantine in certain areas of China [8,9].
The major surface protein 4 (msp4) gene of Anaplasma spp. is highly conserved among many strains [20,27]. It has been demonstrated that PCR amplification of the msp4 gene has a high diagnostic value for the differential detection of A. ovis [20,22,28]. The msp4 gene has also been applied to genetic characterization and phylogenetic studies of Anaplasma spp., thus providing its biogeographic and evolutionary information. Our aim was to investigate the presence of Anaplasma spp. in pupal and adult M. ovinus.

Study areas and M. ovinus collection
In 2013, five M. ovinus specimens were collected during occasional tick sampling in South Xinjiang and were In July 2016, 30 experimental specimens preserved in 70% ethanol were randomly selected from~300 M. ovinus specimens collected from multiple sheep in Yahazhen of Kuqa in Aksu, Xinjiang (1029 m above sea level; 41°44'N, 83°14'E).
In June 2017, over 200 M. ovinus specimens were collected from each of the five sheep in Yahazhen of Kuqa in Aksu, Xinjiang. These M. ovinus specimens were placed in sampling vials with sufficient air and transported immediately to the laboratory for cryopreservation. Ten randomly selected M. ovinus specimens from each sheep and eight simultaneously collected pupal M. ovinus specimens from three sheep were regarded as experimental specimens.
DNA extraction, PCR of the msp4 gene, and sequence analysis The 70% ethanol-preserved M. ovinus specimens were washed twice with distilled water after being washed in a graded series of ethanol solutions with concentrations of 50%, 30% and 10%. The cryopreserved adult and pupal M. ovinus specimens were washed twice with distilled water for 1 h each.
Next, the genomic DNA of M. ovinus was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 (Takara, Dalian, China, catalogue No. 9765). At the last step, the DNA sample was eluted twice with 50 μl of elution buffer, and the resultant 50 μl of genomic DNA was stored at -20°C until use.
The cycling conditions for the msp4 gene amplification with primers MSP4-F and MSP4-R were as follows: initial denaturation at 94°C for 5 min; 40 cycles at 94°C for 30 s, 62°C for 50 s, and 72°C for 1 min; followed by final extension at 72°C for 10 min.
All the amplicons were bidirectionally sequenced on an ABI PRISM TM 3730XL DNA Analyzer (ABI, CarIsbad, America). The sequences were aligned with reference sequences downloaded from GenBank by means of MEGA 7.0 software. The sequences obtained in this study were deposited in the GenBank database under the accession numbers MG283274 and MG564176.

Results
The Sequences of the two taxa obtained in this study having the highest similarity with the msp4 gene sequence of Anaplasma spp. in the GenBank database are listed in Table 1, both of which are A. ovis isolates. The phylogenetic analysis of msp4 confirmed that the obtained Anaplasma sp. was A. ovis (Fig. 2). Additionally, A. ovis isolates XJNJ (MG283274) and XJNJ2 (MG564176) were classified as A. ovis msp4 Genotypes II and III based on A 360 T 366 G 400 C 470 T 522 A 630 C 774 and A 360 T 366 G 400 T 470 T 522 A 630 C 774 , respectively (Fig. 3).

Discussion
The presence of A. ovis DNA in adult and pupal M. ovinus collected in South Xinjiang, China, was confirmed by conventional PCR and sequencing. The sequence variation in the msp4 gene among different A. ovis strains [20] confirmed that two genotypes of A. ovis were detected in this study.
The detection of A. ovis in M. ovinus has been reported previously [15]. Anaplasma ovis has also been discovered in adults of hippoboscid species (Lipoptena cervi), but not in the larvae and pupae [17]. Bartonella [4,7], Arsenophonus and Wolbachia [7] can be transmitted vertically in M. ovinus. Both M. ovinus [4,7,17] and L. cervi mediate vertical transmission of Bartonella [29]. Nevertheless, the vertical transmission of A. ovis via parasites belonging to the family Hippoboscidae (Diptera: Hippoboscoidea) has not been reported. To the best of our knowledge, this study provides the first molecular evidence for the presence of A. ovis DNA in pupal M. ovinus. Additionally, the detection of A. ovis DNA in M. ovinus has not been reported in China. Our study suggests that A. ovis may be transmitted vertically via M. ovinus, and that M. ovinus may serve as a potential vector for A. ovis.
The diseases caused by Anaplasma spp. are a global issue, among which A. ovis causes ovine anaplasmosis. First discovered in sheep in 1912, A. ovis is currently widely distributed in Africa, Europe, Asia, and the USA [24,25]. In China, A. ovis was first found in 1982 in the Xinjiang Uygur Autonomous Region, followed by Liaoning Province. Subsequent studies revealed that A. ovis is widely distributed in China and is particularly prevalent in the northwest region [22,25]. Anaplasma ovis mainly parasitizes sheep, goats, wild ruminants [30,31], cattle [28] and dogs [32]. Recently, an A. ovis variant was detected in a patient, indicating the zoonotic potential of this agent [33]. In addition, some sequences having the highest similarity with the msp4 gene sequence of the two M. ovinus-derived A. ovis isolates in this study were detected in the blood of sheep sampled in Xinjiang in 2012. Taken together, Xinjiang has been seriously infested with A. ovis. It has been confirmed that various ticks, belonging to the genus Ixodes, serve as biological vectors for the transmission of A. ovis in China [22]. Our study confirmed the transmission of A. ovis via M. ovinus in China. Furthermore, currently there are seven  ovis strains by application of the ML method to the msp4 gene sequence data. Evolutionary analyses were conducted in MEGA 7. Nucleotide sequence differences among the msp4 gene sequences from different isolates of A. ovis confirmed seven genotypes [20]. Sequences of our specimens are marked with red circles