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To date, there is no report on the genetic diversity of ticks in these regions. A total of 370 representative ticks from the south and east regions of Kazakhstan (SERK) and Xinjiang Uygur Autonomous Region (XUAR) were selected for molecular comparison. A fragment of the mitochondrial cytochrome c oxidase subunit I (cox1) gene, ranging from 631 bp to 889 bp, was used to analyze genetic diversity among these ticks. Phylogenetic analyses indicated 7 tick species including Hyalomma asiaticum, Hyalomma detritum, Hyalomma anatolicum, Dermacentor marginatus, Rhipicephalus sanguineus, Rhipicephalus turanicus and Haemaphysalis erinacei from the SERK clustered together with conspecific ticks from the XUAR. The network diagram of haplotypes showed that i) Hy. asiaticum from Almaty and Kyzylorda Oblasts together with that from Yuli County of XUAR constituted haplogroup H-2, and the lineage from Chimkent City of South Kazakhstan was newly evolved; and ii) the R. turanicus ticks sampled in Israel, Almaty, South Kazakhstan, Usu City, Ulugqat and Baicheng Counties of XUAR were derivated from an old lineage in Alataw City of XUAR. These findings indicate that: i) Hy. asiaticum, R. turanicus and Ha. erinacei shared genetic similarities between the SERK and XUAR; and ii) Hy. marginatum and D. reticulatus show differences in their evolution.

Background The red fox (Vulpes vulpes) is a geographically widespread predator species, occurring across the northern Hemisphere. It has high veterinary-medical significance, in part owing to its definitive host role in the life cycle of several tapeworm species, including zoonotic ones. However, there have been only few reports on cyclophyllid tapeworm (Cestoda: Cyclophyllidea) infection in red foxes from China. Methods In this study, 263 tapeworms were collected from eight road-killed red foxes in Xinjiang Uygur Autonomous Region (XUAR, northwestern China). The tapeworms were analyzed based on morphological characters and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences. Results 83 Taenia and 180 Mesocestoides tapeworms were identified according to the presence or absence of rostellum, and the number, the length and the shape of the large rostellar hooks. The morphological and molecular analyses revealed that i) 180 Mesocestoides tapeworms, here named as Mesocestoides sp. Vulpes vulpes, showed 99.21% (378/381 bp) identity to Mesocestoides sp. reported from red fox in Mongolia; and ii) 83 Taenia tapeworms belonged to three species. The first Taenia species (n = 16, named as Taenia sp. Vulpes vulpes), based on the length of large rostellar hooks (337–342 µm) and its cox1 sequence, was identified as a potentially novel species, which is phylogenetically close to Taenia laticollis. The second species (n = 54, named as Taenia sp. Vulpes vulpes & Rhombomys opimus), was morphologically similar to Taenia endothoracicus according to the number (n = 52), the length (319–332 µm) and the shape of the large rostellar hooks. This species, infecting three red foxes, shared 100% cox1 sequence identity with Taenia sp. Rhombomys opimus genotype C found previously in great gerbils (Rhombomys opimus) in the same region. The third species (n = 13, named as Taenia polyacantha-like), had shorter large rostellar hooks (178–180 µm) and showed 96.27% (361/375 bp) sequence identity to Taenia polyacantha reported from red fox in Italy. In addition, co-infections with two Taenia species and with Taenia and Mesocestoides species were also demonstrated. Conclusions The “great gerbil-red fox” life cycle of Taenia sp. Vulpes vulpes & Rhombomys opimus, belonging to the mitochondrial lineage of T. endothoracicus, is confirmed. The T. polyacantha-like species and Mesocestoides sp. Vulpes vulpes were found for the first time in red fox in China. Taenia sp. Vulpes vulpes is a potentially novel species, which is close to Taenia laticollis based on its phylogenetic properties. The life cycle of Taenia sp. Vulpes vulpes and Mesocestoides sp. Vulpes vulpes should be further explored.

BackgroundThe Eurasian lynx (Lynx lynx) is a medium-sized wild cat species distributed throughout Eurasia, from Europe to the Far East. There has been no report on Taenia species (Cestoda: Cyclophyllidea) infecting this felid in China.MethodsIn this study, 24 tapeworms were found in two Eurasian lynxes (#1 and #2) in Xinjiang, northwestern China. The tapeworms were identified based on two mitochondrial genetic markers, the cytochrome c oxidase subunit 1 (cox1) and 16S rDNA. This was followed by detailed morphologic characterization.ResultsMolecular and phylogenetic analyses of the cox1 gene revealed that i) a single tapeworm from lynx #2 shared 100% identity with Taenia laticollis genotype C detected in Eurasian lynx in Finland, and ii) the remaining 23 tapeworms, provisionally named here as “Taenia sp. Eurasian lynx”, had two nucleotide substitutions but phylogenetically clustered together. The latter showed only 93.25% sequence identity to T. hydatigena from sheep (Ovis aries) in Slovakia. The scolex morphology is characteristic enough to distinguish “Taenia sp. Eurasian lynx” from other species of its genus.Conclusions “Taenia sp. Eurasian lynx” is a novel tapeworm species found in Eurasian lynx. In addition, T. laticollis was found in this wild felid for the first time in China. The intermediate hosts of T. laticollis and “Taenia sp. Eurasian lynx” should be explored in the near future.

Background Previously, twelve Rickettsia species were identified in ticks, fleas, sheep keds (Melophagus ovinus), bats (Pipistrellus pipistrellus) and a tick-bitten patient in the Xinjiang Uygur Autonomous Region (XUAR) in northwestern China. Here we aimed to molecularly detect rickettsial agents in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks. Methods During 2018–2019, 12 red foxes, one marbled polecat and their ticks were sampled in two counties and a city of the XUAR. The heart, liver, spleen, lung and kidney of these 13 carnivores were dissected, followed by DNA extraction. Hard ticks were identified both morphologically and molecularly. All samples were examined for the presence of rickettsiae by amplifying four genetic markers (17-kDa, gltA, ompA, sca1). Results A total of 26 adult ticks and 28 nymphs (38 Ixodes canisuga, nine Ixodes kaiseri, six Haemaphysalis erinacei and one Dermacentor marginatus) were collected from red foxes, and four Ha. erinacei ticks were removed from the marbled polecat. Analysis of cytochrome c oxidase subunit I (COI) gene sequences indicated that 2–32 nucleotides differed between I. canisuga, I. kaiseri and Ha. erinacei from northwestern China and Europe. Rickettsia raoultii was detected in three red foxes, Candidatus Rickettsia barbariae in a red fox, Rickettsia sibirica in a red fox and a marbled polecat, and R. raoultii in two tick species (I. canisuga and D. marginatus). Conclusions To the best of our knowledge, I. canisuga and I. kaiseri have not been previously reported from red foxes in China. The DNA of R. sibirica and R. raoultii was detected for the first time in the organs of red foxes, and R. sibirica in the organs of a marbled polecat. This is also the first molecular evidence for the presence of R. raoultii in I. canisuga. Our findings expand the range of tick-borne pathogens in wildlife species and associated ticks in China. Graphical Abstract

Babesia species (Apicomplexa: Piroplasmorida) are tick-borne protozoan hemoparasites, which pose a significant threat to domestic animals, wildlife and humans. This study aimed to determine and characterize Babesia species in red foxes (Vulpes vulpes), Asian badgers (Meles leucurus) and their ticks. Blood, heart, liver, spleen, lung, kidney, large intestine and small intestine were collected from 19 wild carnivores (12 red foxes and 7 Asian badgers). All ticks were removed from these animals and identified according to morphological and molecular characteristics. The samples were tested for the presence of Babesia species using the 18S rRNA gene. Molecular analyses showed that the DNA of Babesia vogeli and Babesia vulpes was present in red fox organs/tissues and blood samples. A total of 54 hard ticks (38 Ixodes canisuga, 6 Haemaphysalis erinacei, 9 Ixodes kaiseri and 1 Dermacentor marginatus) were collected from red foxes and 12 (I. kaiseri) from Asian badgers. All ticks were adults. Among them, one I. kaiseri parasiting a red fox contained the DNA of B. vulpes while one I. canisuga was positive for Babesia sp. belonging to the clade "Babesia sensu stricto". Molecular and phylogenetic analyses indicated the presence of a novel genotype, Babesia sp. "badger China". Babesia sp. badger type A and type B from Asian badgers were different from those in European badgers. Co-infection with three Babesia genotypes was found in one Asian badger. This study provides the first data on Babesia infection in red foxes, Asian badgers and their ticks in China. Babesia vogeli was detected for the first time in red foxes in Asia. Co-infection and genetic diversity of Babesia genotypes in Asian badgers were also demonstrated.

Piroplasmosis is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks in Eastern and Southern Kazakhstan (ESK). During 2016 – 2019, adult ticks (at 26 sampling sites in 16 districts of 5 oblasts in ESK) were collected. Tick species were identified according to morphological and molecular characteristics. Two fragments (487 bp and 438 bp) of 18S ribosomal RNA (18S rRNA) were used to determine piroplasm species in representative 698 ticks. The genotype characteristics of Babesia caballi and Theileria equi were further analyzed by longer 18S rRNA gene fragments. A total of 6107 adult ticks (4558 parasitizing ticks and 1549 off-host ticks), including 4665 hard ticks and 1442 soft ticks, were collected from their natural hosts (cattle, horses, sheep, camels, shepherd dogs and hedgehogs) and the surrounding environment, respectively. Among the hard tick species, Dermacentor marginatus (62.59%, 2920/4665) was the most abundant, followed by Hyalomma asiaticum (19.36%, 903/4665) and Hyalomma detritum (9.95%, 464/4665). All soft ticks were identified as Argas persicus. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by Haemaphysalis erinacei and D. marginatus, clustered together with conspecific ticks reported from China. Five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected in 698 representative ticks. Genotype E of T. equi in Almaty, and genotype A of B. caballi in Almaty and South Kazakhstan were identified.

Background The Republic of Kazakhstan, located in Central Asia, ranks as the world’s largest landlocked country, and borders five countries including China. There is a 1783 km long borderline between the South and East regions of Kazakhstan and Xinjiang Uygur Autonomous Region (XUAR, northwestern China). To date, there is no report on the genetic diversity of ticks in these regions. Methods During 2015–2019, a total of 4392 hard ticks (representatives of ten species) were collected from 605 animals (sheep, cattle, camels, dogs and hedgehogs) at 24 sampling sites belonging to 15 districts in southeastern Kazakhstan. After morphological identification, 213 specimens of these ticks were selected for molecular analyses. In addition, 157 hard ticks collected from sheep and camels between 2015 and 2018 in seven districts of XUAR were used for comparison. Following DNA extraction, a fragment of the mitochondrial cytochrome c oxidase subunit I (cox1) gene, ranging from 631 bp to 889 bp, was used to analyze genetic diversity among these ticks. Findings: Phylogenetic analyses indicated that i) five tick species including Hyalomma detritum, Hyalomma asiaticum, Rhipicephalus turanicus, Dermacentor reticulatus and Haemaphysalis erinacei from Kazakhstan clustered together with conspecific ticks from XUAR; ii) the phylogenetic separation of Dermacentor marginatus from Kazakhstan and XUAR was highly supported; and iii) Rhipicephalus sanguineus sensu lato from Alamaty Oblast was more closely related to a specimen from Iran than to that from XUAR. The network diagram of haplotypes showed that iv) Hy. asiaticum from Almaty and Kyzylorda (Kazakhstan) together with that from Yuli County of XUAR constituted an ancestral haplogroup; and v) three lineages of Rh. turanicus (from Israel, Almaty and South Kazakhstan, as well as from Usu city, Ulugqat and Baicheng Counties of XUAR) might have originated from an ancestral lineage in Alataw city, XUAR. Conclusions These findings indicate that: (i) mitochondrial lineages of some tick species are shared between southern, eastern regions of Kazakhstan and northwestern China; (ii) common evolutionary origin of Hy. asiaticum and Rh. turanicus in these regions might be attributed to historical international trade and movements of wildlife; and (iii) certain tick species show clear differences between Kazakhstan and XUAR, either in terms of abundance (e.g. Hy. scupense, Hy. marginatum) or exhibiting a phylogenetic split between these regions (relevant to D. marginatus).

Background: Piroplasmosis, caused by Theileria and Babesia species, is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks from different host species in Kazakhstan. Method: We collected adult ticks at 26 sampling sites in 16 districts in the East and South regions of Kazakhstan during 2016 - 2019. Tick species were identified according to morphological and molecular characteristics. Two fragments of the 18S ribosomal RNA (18S rRNA) were used to determine the species of piroplasms. The genotype characteristics of Babesia caballi and Theileria equi were analyzed further. Results: A total of 6107 adult ticks were collected, including 2920 Dermacentor marginatus, 1442 Argas persicus, 903 Hyalomma asiaticum, 464 Hyalomma scupense, 225 Hyalomma anatolicum, 122 Rhipicephalus turanicus, 16 Haemaphysalis erinacei and 15 Dermacentor reticulatus. In these ticks five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected. Theileria equi and Babesia caballi belonged to their E and A genotype, respectively.Conclusions: Apart from Dermacentor marginatus, three Hyalomma species predominate among ticks infesting herbivorous livestock in East and South regions of Kazakhstan. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by D. marginatus and Ha. erinacei, were clustered together with conspecific ticks reported from China. Babesia occultans was found for the first time in D. marginatus in Kazakhstan. Altogether five piroplasm species were detected, indicating that enhanced control measures are necessary to prevent piroplasm infection of domestic animals in this region.

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. Electronic supplementary material The online version of this article (10.1007/s10493-018-00337-1) contains supplementary material, which is available to authorized users.

Background Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. To analyze the clonal expression and immunogenicity of the Ye Mule Aries sheep inhibin gene, total RNA was extracted from sheep ovarian tissue and used as a template to generate a eukaryotic expression vector and study inhibin immunogenicity. Methods Primers were designed to amplify the inhibin A gene via polymerase chain reaction and the amplified product was cloned between the ScalI and EcoRI restriction sites of the expression vector pEGFP-N1 to construct a recombinant plasmid, pEGFP-INHα. Following the validation of successful cloning, the pEGFP-INHα plasmid was transfected into BHK cells to verify expression in eukaryotes and subsequently utilized as an antigen in rabbits. Rabbits were tested for anti-inhibin antibodies and serum follicle-stimulating hormone (FSH) concentrations. Results The analysis of the INHα gene sequence revealed that INHα is 1109 bp long and is translated to an approximately 40 KDa protein. Bioinformatics approach indicated that the INHα gene is highly conserved between organisms. Immunization with the eukaryotic expression vector, pEGFP-INHα, which expresses the INHα gene elicited immune response and generatigeneration on of anti-INHα antibody. The antibody had a significant regulatory effect on the serum concentration of FSH in rabbits and led to higher levels of FSH, indicating increased ovary function. Conclusions The present work resulted in a successful construction of eukaryotic expression plasmid pEGFP-INHα and verified the immunogenicity of this highly conserved protein. Further, the expression of pEGFP-INHα was shown to have a significant impact on the secretion of FSH, indicating a potential regulatory role in ovarian function. In conclusion, our current findings can serve as a working model for studying the effect of INHα on the breeding performance of Ye Mule Aries sheep, providing a novel strategy to improve their reproduction rates.

In this study, two road-killed marbled polecats (Vormela peregusna) were molecularly analysed for tick-borne pathogens. Rickettsia raoultii, “Candidatus Rickettsia barbariae” and a novel Babesia genotype have been identified, for the first time in marbled polecat. DNA of this Babesia sp. genotype was also present in four out of 15 Haemaphysalis erinacei ticks collected from the Babesia PCR-positive marbled polecat. Results of this study suggest that marbled polecats may serve as reservoirs for these bacteria and protozoans. Electronic supplementary material The online version of this article (10.1186/s13071-018-3033-z) contains supplementary material, which is available to authorized users.

Table S1. Information for the sequences from the GenBank database used in Fig. 1. (DOCX 18 kb)