Vector-Borne Pathogens with Veterinary and Public Health Significance in Melophagus ovinus (Sheep Ked) from the Qinghai-Tibet Plateau

Melophagus ovinus (sheep ked) is a hematophagous ectoparasite that mainly parasitizes sheep. In addition to causing inflammation, wool loss, and skin damage to the animal hosts, M. ovinus also serves as a vector for a variety of pathogens and is highly likely to participate in the life and transmission cycle of pathogenic organisms. Herein, we investigated the presence and molecular characterization of vector-borne pathogens in M. ovinus from Qinghai-Tibet Plateau, China. A total of 92 M. ovinus pools collected from the Qinghai province of China were screened for the presence of selected vector-borne pathogens. The overall positive rate of A. ovis, A. bovis, A. phagocytophilum, and T. ovis in M. ovinus was 39.1%, 17.4%, 9.8%, and 89.1%, respectively. All of the samples were negative for Border disease virus (BDV), other Anaplasma species, Babesia spp., Rickettsia spp., and Borrelia spp. Co-infection of different Anaplasma species and T. ovis occurred in 51.2% of all samples with T. ovis. The positive rates of A. ovis, A. bovis, and A. phagocytophilum in different regions and altitudes of the sampling sites were significantly different. Sequence and phylogenetic analysis of target genes confirmed their identity with corresponding pathogens. Our results elucidate the occurrence and molecular characterization of Anaplasma spp. and Theileria spp. in M. ovinus, which could act as potential zoonotic reservoirs. To the best of our knowledge, this is the first report of the detection of A. bovis and A. phagocytophilum DNA in M. ovinus. This study gives the first extensive molecular survey of vector-borne pathogens with veterinary and public health significance in M. ovinus from the Qinghai-Tibet Plateau, China.


Introduction
Melophagus ovinus (sheep ked) belongs to the family Hippoboscidae (Diptera: Hippoboscoidea) and is a blood-feeding ectoparasite of livestock and wild animals, including sheep, goats, rabbits, dogs, Tibetan antelope, European bison, and red foxes, and has also been found in humans [1][2][3]. The life cycle of sheep ked comprises the larva, pupa, and wingless adult stages, and all life stages of this ectoparasite occur on the host. After mating, the females produced single larvae every 6-8 days that were attached to the host wool and molded into the puparial stage within 6-12 h. It takes the pupae 19-30 days to develop into an adult [3]. Adults are commonly circulated among the animals and transferred from ewes to their offspring by direct contact. Melophagus ovinus (M. ovinus) is reported to cause inflammation, wool loss, skin damage, and reductions in weight gain of sheep and has significant economic effects in the sheep industry [4,5]. Of the sheep studied, 61-81% of sheep were infested with M. ovinus [3]. Melophagus ovinus is broadly distributed in Africa, Europe, Oceania, North America, and Asia [5]. In China, M. ovinus has recently been reported to parasitize sheep and Tibetan antelopes in Tibet, Xinjiang, Qinghai, and Gansu, and was also detected in imported sheep and sheep wool in certain areas of China [6][7][8].
Melophagus ovinus serves as potential vectors of a variety of pathogens and has been reported to be responsible for the transmission of pathogenic organisms such as helminths, protozoa, bacteria, and viruses due to their blood-feeding behavior towards hosts [1,9]. M. ovinus was reported to mechanically transmit the Bluetongue virus in sheep [2]. Additionally, previous studies showed that M. ovinus may be a vector for Bartonella schoenbuchensis and B. chomeli in the USA [10], Anaplasma ovis in Hungary [11], Acinetobacter spp. in Ethiopia [12], and Bartonella in Central Europe [2]. Chu et al. [6] reported Borrelia burgdorferi sensu lato in sheep keds in Tibet, China. Recently, in China, Anaplasma ovis [13], Rickettsia raoultii and R. slovaca [5], Theileria ovis [9], and Border disease virus (BDV) [14] have also been detected in M. ovinus in the Xinjiang Uygur Autonomous Region of northwestern China.
The Qinghai province is one of the traditional animal husbandry bases in China and a small number of reports have recorded the presence of M. ovinus in the region [5]. However, very little is known about the occurrence of arthropod-borne pathogens in M. ovinus from the Qinghai. Given the veterinary and public health significance of M. ovinus, the objective of the present study was to investigate the presence of vector-borne pathogens in M. ovinus from the Qinghai-Tibet Plateau of China.

Results
A total of 92 M. ovinus pools were screened for the presence of selected vector-borne pathogens. Of the 92 samples tested, 46 (50.0%) pools were positive for one or more Anaplasma species. The infection rates were 39.1%, 17.4%, and 9.8% for Anaplasma ovis (A. ovis), Anaplasma bovis (A. bovis), and Anaplasma phagocytophilum (A. phagocytophilum) in M. ovinus, respectively (Table 1). Importantly, A. bovis, and A. phagocytophilum were detected in M. ovinus for the first time. A total of 82 pools (89.1%) were positive for piroplasm infections, and all of which belonged to Theileria ovis (T. ovis). No positive results were obtained for other tested pathogens, including BDV, Anaplasma centrale, Anaplasma platys, Anaplasma capra, Anaplasma marginale, Babesia spp., Rickettsia spp., and Borrelia spp. Mixed infections of both the T. ovis and Anaplasma species accounted for 51.2% (42/82) of all samples with the T. ovis. Anaplasma ovis co-infections with A. bovis and A. phagocytophilum accounted for 26.1% (12/46) and 2.2% (1/46) of Anaplasma species infections, respectively. Sequence analysis of the msp4 sequences of A. ovis (sequence similarity 100%), 16S rRNA sequences of A. bovis (sequence similarity 99.9-100%) and A. phagocytophilum (sequence similarity 100%), and 18S rRNA gene sequences of T. ovis (sequence similarity 99.9-100%) confirmed their identity with corresponding pathogens by using BLASTn search. Phylogenetic analysis of the msp4 sequences represented showed that the MW147462 sequence was classified as A. ovis Genotypes II based on nucleotide mutation sites (A 360 T 366 G 400 ). Sequence MW142385 of A. phagocytophilum was classified into cluster I, and sequence MW142384 of A. bovis was identical with strains isolated from sheep (MT036513), tick (KC311345), horse (MK028574), and deer (KJ659040) ( Figure 1A-C). The phylogenetic analysis of the 18S rRNA gene confirmed that the detected piroplasm (MW142379) was T. ovis ( Figure 1D).  Risk factors including M. ovinus gender, the region, and altitude of the sampling sites were used as variables for statistical analysis of the infection patterns of Anaplasma spp. and Theileria spp. occurrence (Table 2). For the occurrence of A. ovis, A. bovis, and A. phagocytophilum, significant differences between locations have been observed. Melophagus ovinus collected in Haidong had a higher risk than other M. ovinus in Golog and Yushu to be infected with A. ovis (p = 0.01) and A. bovis (p = 0.003), while A. phagocytophilum infection rate in M. ovinus collected in Yushu was significantly higher than Haidong and Golog (p = 0.011). Melophagus ovinus collected at 3000 m areas was at higher risk of being infected with A. ovis (p = 0.033) and A. bovis (p = 0.007) than in M. ovinus collected at altitudes of 3800 m and 4100 m. Besides, the results showed no significant difference in gender.

Discussion
To date, few publications have described the distribution and prevalence of vectorborne pathogens in M. ovinus from the Qinghai-Tibet Plateau, China [5,6]. As the traditional animal husbandry base, epidemiological investigations into vector-borne pathogens with veterinary and public health significance in Qinghai are of particular importance. Anaplasma spp. occurrence in M. ovinus demonstrated a wide distribution of A. ovis, A. bovis, and A. phagocytophilum in the Qinghai-Tibet Plateau, China. A. ovis has been considered as the etiological agents of anaplasmosis of domestic ruminants and it has been widely detected in sheep, goats, wild deer, and many tick species around the world [15,16]. In previous reports, all sheep keds (100%, 81/81) were found to harbor A. ovis in Hungary [11] and 28 specimens (including five pupal specimens) (31.8%, 28/88) collected in 2016 and 2017 in Xinjiang, China tested positive for A. ovis [13]. The positive rate (39.1%) and genetic characteristic (Genotype I) of A. ovis in M. ovinus in this study concurred with other reports published in Xinjiang.
To the best of our knowledge, this is the first molecular evidence of A. bovis and A. phagocytophilum in M. ovinus over the world. Anaplasma bovis mainly affects cattle with fever, progressive anemia, and even death, and the subclinical infections of this agent have also been found in small mammals and ruminants, indicating the reservoir competence of those animals for A. bovis [17]. Besides, A. bovis can be found in many tick species (Haemaphysalis longicornis, Haemaphysalis lagrangei, Haemaphysalis concinna, and Rhipicephalus evertsi, etc.) in Asia, Europe, and Africa [16,18]. We detected A. bovis with a positive rate of 17.4% in M. ovinus for the first time, which indicated that M. ovinus may be the potential reservoirs or maintenance hosts of this agent. Among the Anaplasma species detected, A. phagocytophilum is an emerging zoonotic pathogen of human and animal granulocytic anaplasmosis and can be transmitted to a wide range of mammals including humans, ruminants, horses, cats, dogs, rodents, birds, and reptiles through the bite of ticks [19]. In the present study, this is the first time that A. phagocytophilum DNA has been detected in M. ovinus using the molecular biological method. Statistical analysis indicated that Aanaplasma spp. infections showed significant correlations with the region and altitude of the sampling sites and the co-infection. Our results expand the potential vector spectrum of A. bovis and A. phagocytophilum and emphasize the veterinary and public health significance of M. ovinus.
Parasitic protists of the genus Theileria, especially T. annulata, T. sergenti, and T. hirci, are the causative agent of Theileriosis and have a wide geographical and host-species distribution. Among the Theileria species, Theileria ovis mainly causes benign theileriosis in sheep and goats, which is easily overlooked [20]. Theileria ovis are distributed widely in Asia, Europe, and Africa. In China, T. ovis has mainly been reported in animal, tick, and sheep keds from Xinjiang [9,21], Inner Mongolia [22], Qinghai [23], Sichuan [24]. Several reports have recently shown that there are two species of Theileria spp. (T. ovis and T. luwenshuni) in M. ovinus [9,24]. Historically, hard ticks were considered as the only and essential vector for Theileria spp. Our findings and similar studies conducted by Zhao [9] expand the potential vector spectrum of T. ovis. Herein, a high positive rate (89.1%, 82/92) of T. ovis DNA was demonstrated in M. ovinus in the present study, but this needs to be confirmed through more testing. Many factors including biogeography, the season of sample collection, number of samples, etc., may contribute to the differences between investigations of pathogenic organisms in M. ovinus in other regions in China or other countries.
Vector-borne pathogens including Anaplasma species, BDV, Babesia spp., Rickettsia spp., and Borrelia spp. cause economic losses in the livestock industry and pose a risk to humans. Although these infectious agents were negative in this study, some of these pathogens were found in tick, yak, and Tibetan sheep samples (unpublished data), implying that this region tends to have a higher risk of vector-borne diseases. Future studies should systematically screen for the presence of potential animal as well as human pathogens in M. ovinus.
We demonstrated the occurrence of A. ovis, A. bovis, and A. phagocytophilum, and T. ovis with veterinary and medical significance in M. ovinus in Qinghai, China. A. bovis and A. phagocytophilum was found for the first time and the present study extended the spectrum of pathogens potentially present in M. ovinus. The occurrence of these pathogens in M. ovinus may be a threat to animal and public health in the Qinghai-Tibet Plateau, China. Future investigations are warranted to elucidate the genetic diversity of vectorborne pathogens in M. ovinus and the role of M. ovinus as the specific biological vectors of some pathogens.

Study Sites and Sample Collection
Adult sheep keds (n = 276) were collected at four sites: Xunhua, Haidong (n = 24, altitude 3000 m, 35 • 39 N 102 • 41 E), Maqin, Golog (n = 24, altitude 3800 m, 35 • 2 N 99 • 12 E), Dari, Golog (n = 24, altitude 4100 m, 33 • 43 N 99 • 38 E), and Zhiduo, Yushu (n = 24, altitude 4100 m, 33 • 37 N 95 • 58 E) during June 2020 in Qinghai province, China (Figure 2). After collection, sheep keds were shipped into the laboratory in cooled flasks and pooled (n = 92, three adults of the same sex collected from the same sheep were pooled) before being frozen at −80 • C until testing. Morphological studies ( Figure 2) and 18S rRNA gene sequence analysis (data not shown) confirmed that the collected samples belong to sheep keds. The study was conducted in compliance with the ethical policies of the journal and the rules of the ethics committee of the Institute of Zoology, Chinese Academy of Sciences.

Nucleic Acid Extraction and PCR Amplification
All samples were sterilized with 70% ethanol and distilled water and were mechanically disrupted in 200 µL of PBS. Genomic DNA and RNA were extracted from 100 µL of the homogenate with the commercially TIANamp Genomic DNA Kit (TIANGEN BIOTECH (BEIJING) CO., LTD, Beijing, China) and Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was synthesized using the GoScript Reverse Transcription System and 5 -UTR of BDV was amplified according to the Access RT-PCR System (Promega, Madison, WI, USA) [14]. Nested PCR-based amplification was employed for the detection of A. bovis, A. phagocytophilum, A. centrale, A. platys, and A. capra based on the 16S rRNA gene and the citrate synthase (gltA) gene, and conventional PCR was used for detection of A. ovis and A. marginale based on the msp4 gene, as previously described [16,[25][26][27]. For piroplasm (Theileria spp. and Babesia spp.) detection, all samples were screened using nested PCR assays targeting the 18S rRNA gene [27]. Other vectorborne bacteria including Rickettsia spp. and Borrelia spp. were also detected [28][29][30] and the PCR primers and cycling conditions are shown in Table 3. The DNAs extracted from the domestic animals and ticks in Qinghai infected with A. ovis, A. bovis, A. phagocytophilum, Theileria ovis, Theileria sinensis, and Candidatus Rickettsia jingxinensis were used as positive controls. The PCR products were detected by 1% agarose gel electrophoresis with M5 Hipure Next III Gelred (Mei5 Biotechnology Co., Ltd., Beijing, China) stained.

Sequencing and Phylogenetic Analysis
The PCR products from positive samples were sequenced at BGI Sequencing (Beijing, China) and subjected to BLAST searches for nucleotide sequence analysis and alignments. Phylogenetic trees were constructed using the neighbor-joining method executed with Kimura 2-parameter model in MEGA X. Bootstrap values were assessed with 1000 bootstrap replicates. The representative nucleotide sequences of this study have been deposited in the GenBank database under accession number MW147462 for A. ovis, MW142384 for A. bovis, MW142385 for A. phagocytophilum, and MW142379 for T. ovis.

Data Analysis
The data were grouped into three variables in terms of sheep keds gender and the region and the altitude of the sampling sites. Differences in infection rates of each group were statistically calculated using the Chi-square test in SPSS 25.0. A p-value of <0.05 was considered significant.