Haemaphysalis Flava Research Articles

Molecular Cloning and Expression Analysis of Enolase in the Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus is the main blooding-sucking ectoparasite of bovines and is regarded as important vectors of animal diseases such as Babesiosis. Mining protective antigens of R. microplus to develop antitick vaccine is the most potential tick control strategy. In this study, the specific primers were designed according to the conserved nucleotide sequence of enolase gene in Haemaphysalis flava, Ixodes ricinus, and Ornithodoros moubata. The fragment of enolase gene was obtained by PCR using cDNA template from fully engorged female R. microplus. The full-length cDNA of enolase gene was amplified using rapid amplification of cDNA ends (RACE). Expression pattern of enolase gene in different tissues of R. microplus was analyzed by real-time quantitative PCR (qRT-PCR). Results showed that the full-length enolase cDNA containing 2052 bp was obtained successfully. The complete cDNA included an ORF of 1305 nucleotides encoding a protein of 434 amino acids. The enolase exhibited 85.0% amino acid identity to the enolase of H. flava, 81.1% to I. ricinus enolase, and 81.3% to O. moubata enolase. qRT-PCR analysis indicated that the enolase had the highest expression in the salivary gland of R. microplus.

Characterization of AV422 from Haemaphysalis flava ticks in vitro.

Ticks are hematophagous ectoparasites and cause a major public health threat worldwide. Development of anti-tick vaccines is regarded to be an optimal alternative for tick control. AV422, a unique protein in ticks, is secreted into hosts during blood-feeding, but its roles are not confirmed in Haemaphysalis flava ticks. We retrieved a gene fragment encoding AV422 from a transcriptome dataset of H. flava, and based on it, we reconstructed the full length of AV422 from H. flava (Hf-AV422) by rapid amplification of cDNA ends. Expression profiles of Hf-AV422 in whole ticks and organs of different engorgement levels were determined by qPCR. Then its opening reading frame (ORF) was expressed in Escherichia coli strain BL21 (DE3). The prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) assays were conducted to test anticoagulant activities of the purified recombinant protein (rHf-AV422). The full length of AV422 was 1152bp. Hf-AV422 showed to be conserved as indicated by multiple sequence alignment. Expression of Hf-AV422 was significantly higher in salivary glands and cuticles than in ovaries. Its expression in whole ticks decreased during engorgement with the highest levels in 1/4 engorged ticks. rHf-AV422 prolonged PT, APTT and TT when incubated with rabbit plasma. Our data demonstrated that Hf-AV422 is a conserved salivary protein with anticoagulant activity. Further studies are needed to test in detail its functional properties to ensure it an adequate antigen candidate for the development of broad-spectrum vaccines against ticks.

Real-time PCR biochip for on-site detection of Coxiella burnetii in ticks

BackgroundQ fever, a zoonosis caused by Coxiella burnetii, has adverse effects on public health. Ticks are vectors of C. burnetii and they contribute to the transmission of the pathogen. A tool for rapid, sensitive, and accurate detection of C. burnetii from ticks is important for the prevention of Q fever.MethodsUltra-rapid real-time PCR (UR-qPCR) as a chip-based real-time PCR system was developed for the detection of C. burnetii from ticks. The UR-qPCR system was established and evaluated for the rapidity, sensitivity, and specificity of C. burnetii detection.ResultsC. burnetii was detected using UR-qPCR from 5644 larval, nymphal, and adult ticks from 408 pools collected from livestock and epidemiologically linked environments in two provinces, Gangwon and Jeju, in Korea. Ticks from three species were identified; Haemaphysalis longicornis accounted for the highest number, present in 333 of 408 pools (81.62%), followed by Haemaphysalis flava in 62 pools (15.19%) and Ixodes nipponensis in 13 pools (3.19%). The rapidity and sensitivity of PCR detection was demonstrated with the sufficient amplification and detection of approximately 56 copies of C. burnetii DNA with only 20 min of PCR amplification. The kappa value for the diagnostic agreement between UR-qPCR and stationary qPCR was in perfect agreement (κ = 1). PCR detection and sequencing indicated that C. burnetii was present in 5 of the 408 pools (1.23%), in which four pools contained H. longicornis and one pool contained H. flava. The infection rates of C. burnetii in the tick pools collected from Gangwon and Jeju Provinces were 1.70% and 0.58%, respectively. Phylogenetic analysis indicated a close relationship between the detected C. burnetii and those originating from goats, humans, and ticks in different countries, such as the USA, France, Germany, and Serbia.ConclusionsThe methods described in this study could be important for the prevention and control of Q fever in the two provinces. The UR-qPCR, with its features of mobility, sensitivity, and rapidity, is helpful for constructing early alert systems in the field for C. burnetii in ticks and could help alleviate the transmission of and economic damage due to Q fever.Graphical

De novo transcriptome sequencing and comparative profiling of the ovary in partially engorged and fully engorged Haemaphysalis flava ticks.

Haemaphysalis flava is the vector of several pathogens and has medical and veterinary importance. Transcriptome information of the ovary of H. flava is unavailable and limits understanding of its molecular basis of reproduction. We studied the ovary transcriptome of partially engorged and fully engorged H. flava using high-throughput RNA sequencing technology. A total of 53,025,360 and 57,942,890 clean reads were obtained with 7.95GB and 8.69GB clean bases in partially engorged ticks (PETs) and fully engorged ticks (FETs), respectively. The clean reads were assembled into 138,711 unigenes. A total of 72,043 unigenes (51.93%) were annotated and 66,668 unigenes (48.07%) were unknown. A total of 38,487 differentially expressed genes (DEGs) were found between PET and FET with 19,031 upregulated genes and 19,456 downregulated genes. The RNA-seq results were validated by qRT-PCR, including six upregulated genes and three downregulated genes. Some unigenes coding for nutrient transporters, proteases, and protease inhibitors were found and analyzed. This study was the first time to perform the transcriptome sequences of the ovary of partially engorged and fully engorged H. flava. The results can benefit the understanding of the molecular basis of ovary maturation and oogenesis of the H. flava and boost the development of the strategies for control of H. flava.

Molecular detection of Rickettsia raoultii, Rickettsia tamurae, and associated pathogens from ticks parasitizing water deer (Hydropotes inermis argyropus) in South Korea

Most defined Rickettsiales, which can be transmitted by ticks, are known to be important zoonotic pathogens. Some of these pathogens can cause severe diseases in humans, including anaplasmosis, rickettsioses, and ehrlichiosis. Previous studies in South Korea have investigated tick-borne pathogens (TBPs) residing in ticks found on grassy vegetation and animals. However, there is limited phylogenetic information on TBPs in ticks parasitizing Korean water deer (KWD; Hydropotes inermis argyropus). This study evaluated the prevalence, risk factors (regions, tick stages, and tick species), and coinfections of TBPs in ticks parasitizing KWD. Were collected a total of 283 hard ticks, including Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis from KWD in South Korea from 2013 to 2017. In 173 tested tick pools, genes for seven TBPs, namely Rickettsia raoultii (20 %), Rickettsia tamurae (1 %), Candidatus Rickettsia longicornii (31 %), Ehrlichia canis (3 %), Anaplasma capra (3 %), Anaplasma bovis (2 %), and Anaplasma sp. (1 %), were detected. The unidentified Anaplasma sp. isolates revealed a 98.4 %–99.3 % sequence identity with Anaplasma sp. in GenBank sequences obtained from ticks. To the best of our knowledge, this is the first study to report the presence of the emerging human pathogen R. tamurae in South Korea. These results should increase awareness about the need for continued development of epidemiological control measures, and medical and veterinary communities must be informed of their high infection potential and clinical complexity in humans.

Survey on tick distribution and tick-borne pathogens in Daejeon and adjacent areas in South Korea

Ticks (Ixodidae, also known as hard ticks) as principal vectors of zoonotic diseases such as severe fever with thrombocytopenia syndrome (SFTS), Lyme borreliosis, relapsing fever, anaplasmosis, ehrlichiosis, babesiosis, coxiellosis (Q fever), and tularemia pose a major public health threat. This study was conducted to identify the distribution profile of ticks and tick-borne pathogens in Daejeon and the adjacent areas in South Korea, where no such epidemiological study has been conducted. From April to October 2019, 16,765 ticks were collected from three genera and four species: Haemaphysalis longicornis (n = 14,949; 89.2 %), Haemaphysalis flava (n = 987; 5.9 %), Ixodes nipponensis (n = 828; 5.0 %), and Amblyomma testudinarium (n = 1; 0.01 %). Homogenized samples of ticks were screened by real-time reverse transcription polymerase chain reaction (PCR), real-time PCR, and PCR for the presence of the following tick-borne pathogens: SFTS virus (SFTSV), Borrelia spp., Babesia microti, Coxiella burnetii, Francisella tularensis, Anaplasma phagocytophilum, and Ehrlichia spp. As a result, SFTSV (2 cases), Borrelia spp. (32 cases), and Babesia microti (7 cases) were detected. The findings of this study will contribute to the prevention and management of tick-borne zoonoses.

Ecological traps and boosters of ixodid ticks: The differing ecological roles of two sympatric introduced mammals

The raccoon (Procyon lotor) and masked palm civet (Paguma larvata) are introduced species in Japan and have become abundant in human-inhabited environments. We surveyed tick infestations and tick ingestion by introduced raccoons and masked palm civets captured in Hayama, Kanagawa Prefecture, Japan between November 2018 and January 2020. We collected ticks from the body surface of animals and tick capitula from the gastrointestinal contents. We collected 18,357 ticks identified as Haemaphysalis flava, Haemaphysalis megaspinosa, Haemaphysalis longicornis, Ixodes ovatus, Ixodes tanuki, and Amblyomma testudinarium from 58 of 60 raccoons and 152 ticks, identified as H. flava and I. tanuki, from 16 of 41 masked palm civets. Furthermore, we obtained 16 capitula from 12 % of raccoons and 106 capitula from 63 % of masked palm civets. Raccoons harbored a greater number of ticks (all stages of H. flava and adult I. tanuki) compared with masked palmed civets, whereas the latter species ingested a greater number of nymphal and larval ticks. The results of this study extend our understanding of the ecological roles of two introduced wildlife species. The raccoon may act as an ecological booster, thereby increasing the success rate of bloodmeals and reproduction in ticks. In contrast, the masked palm civet may act as an ecological trap by effectively grooming to remove ticks and prevent bloodmeals.

Isolation of Severe Fever with Thrombocytopenia Syndrome Virus from Various Tick Species in Area with Human Severe Fever with Thrombocytopenia Syndrome Cases.

Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus, generally called SFTS virus (SFTSV), is an emerging zoonosis in East Asia. In Japan, 50-100 cases of SFTS have been reported each year since the first case was reported in 2013. SFTS is a tick-borne infectious disease, and SFTSV has been isolated from ticks in China and South Korea. Haemaphysalis longicornis and Amblyomma testudinarium are considered the primary vectors in Japan. However, the other tick species seldom feeding on humans might also play an important role in maintaining the virus in nature. In this study, we collected ticks on vegetation around the location where two SFTS patients were estimated to have been infected in Miyazaki Prefecture, Japan, isolated live SFTSV, and performed a phylogenetic analysis. A total of 257 ticks were collected, and SFTSV RNA was detected in 19.5% (9/46) of tick pools. A total of 10 infectious SFTSVs were successfully isolated from A. testudinarium, Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis hystricis, and Haemaphysalis megaspinosa. Furthermore, the whole viral sequences isolated from ticks were highly homologous to sequences isolated from SFTS patients in the same sampling area in the past. These results suggest that SFTSVs are maintained in these tick species in the sampling area and sporadically transmitted to humans. Surveillance of SFTSV in ticks provides important information about the risk of incidental transmission to humans.

Diversity and distribution of ticks in Niigata prefecture, Japan (2016–2018): Changes since 1950

We performed tick surveys in all regions (Kaetsu, Chuetsu, Joetsu, and Sado) of the Niigata prefecture, Japan. A total of 105 field surveys were done from 2016 to 2018 in 41 sites, from north to south, in the prefecture. All 4806 ticks collected were identified and classified by species, sex, and developmental stage. Twelve species were recorded: Dermacentor taiwanensis, Haemaphysalis flava, Haemaphysalis hystricis, Haemaphysalis japonica, Haemaphysalis longicornis, Haemaphysalis megaspinosa, Ixodes ovatus, Ixodes nipponensis, Ixodes persulcatus, Ixodes monospinosus, Ixodes columnae, and Ixodes turdus. The major tick species in Niigata prefecture were H. flava, H. longicornis, and I. ovatus and they comprised 93.4% of all samples. These three species have one generation per year. Climatic and anthropogenic factors may be involved in the substantial change of the endemic species composition from a previous tick survey (1959) in the Niigata prefecture. These factors include increasing temperatures, introduction of new hosts such as the wild boar, highway construction, and a rural exodus facilitating animal migration and reproduction. Tick hosts suitable for the transmission of Japanese spotted fever, Lyme borreliosis, and SFTS occur in Niigata prefecture. Heightened awareness of these three tick-borne diseases is needed for preparation and disease prevention.

Spotted fever group rickettsiae (SFGR) detection in ticks following reported human case of Japanese spotted fever in Niigata Prefecture, Japan

Japanese spotted fever, a tick-borne disease caused by Rickettsia japonica, was firstly described in southwestern Japan. There was a suspicion of Rickettsia japonica infected ticks reaching the non-endemic Niigata Prefecture after a confirmed case of Japanese spotted fever in July 2014. Therefore, from 2015 to 2017, 38 sites were surveyed and rickettsial pathogens were investigated in ticks from north to south of Niigata Prefecture including Sado island. A total of 3336 ticks were collected and identified revealing ticks of three genera and ten species: Dermacentor taiwanensis, Haemaphysalis flava, Haemaphysalis hystricis, Haemaphysalis longicornis, Haemaphysalis megaspinosa, Ixodes columnae, Ixodes monospinosus, Ixodes nipponensis, Ixodes ovatus, and Ixodes persulcatus. Investigation of rickettsial DNA showed no ticks infected by R. japonica. However, three species of spotted fever group rickettsiae (SFGR) were found in ticks, R. asiatica, R. helvetica, and R. monacensis, confirming Niigata Prefecture as a new endemic area to SFGR. These results highlight the need for public awareness of the occurrence of this tick-borne disease, which necessitates the establishment of public health initiatives to mitigate its spread.

Detection of Multiple Intracellular Bacterial Pathogens in Haemaphysalis flava Ticks Collected from Hedgehogs in Central China.

Tickborne intracellular bacterial pathogens including Anaplasma, Coxiella burnetti, Ehrlichia, and Rickettsia cause emerging infectious diseases worldwide. PCR was used to amplify the genes of these pathogens in Haemaphysalis flava ticks collected from hedgehogs in Central China. Among 125 samples including 20 egg batches, 24 engorged females, and 81 molted male and female adult ticks, the DNA sequences and phylogenetic analysis showed that the minimum infection rate of the ticks was 4% (5/125) for A. bovis, 3.2% (4/125) for C. burnetti, 9.6%, (12/125) for E. ewingii, and 5.6% for Rickettsia including R. japonica (3.2%, 4/125) and R. raoultii (2.4%, 3/125), respectively. The prevalence of these pathogens was significantly higher in dead engorged females (83.3%, 20/24) than in eggs (5%, 1/20) and molted ticks (8.6%, 7/81). Our study indicated that H. flava ticks could be infected with multiple species of tickborne pathogens including Anaplasma, C. burnetti, Ehrlichia, and Rickettsia in Central China, and the prevalence of these pathogens was reduced during transovarial and transstadial transmission in ticks, suggesting that ticks may not be real reservoirs but only vectors for these tickborne pathogens.

Analysis of <i>Trypanosoma</i> sequences from <i>Haemaphysalis flava</i> (Acari: Ixodidae) and <i>Tabanus rufidens</i> (Diptera: Tabanidae) collected in Ishikawa, Japan

Trypanosoma are known to be a diverse group of parasites that infect animals belonging to all classes in the subphylum Vertebrata and are important pathogens that affect human and animal health. Although many trypanosomatids have been found in mammals and birds in Japan, information regarding their invertebrate host is currently lacking. During our virome analyses of ticks and horse flies, several trypanosoma-like sequences were found. Further sequence characterization and PCR-based screening revealed trypanosomatids termed Trypanosoma sp. 17ISK-T2 and 17ISK-T22 in the nymphs of Haemaphysalis flava, and T. theileri-like sequences in Tabanus rufidens. These results indicate that virome analysis by next-generation sequencing (NGS) can also be used as a tool for protozoan detection from arthropods. Further investigations will assist in understanding the diversity and transmission dynamics of these parasites in Japan.

Detection of Tick-Borne Pathogens in Ticks from Dogs and Cats in the Yamagata Prefecture of Japan in 2018.

Companion animals can become infected with tick-borne diseases (TBDs) becoming a reservoir for human transfer, thereby damaging human health. To evaluate whether companion animals are infested with ticks harboring human TBD pathogens, we detected TBD pathogens in ticks collected from dogs and cats brought to animal hospitals in the Yamagata prefecture of Japan. An investigation of 164 adult ticks collected from 88 dogs and 41 cats between March and July 2018 revealed that this region was dominated by three tick species, Ixodes ovatus (n = 95, 57.9%), Ixodes nipponensis (n = 37, 22.6%) and Haemaphysalis flava (n = 10, 6.1%). To evaluate their pathogenic potential, we went on to test each tick for spotted fever group rickettsiae, Lyme disease borreliae, relapsing fever borreliae, tick-borne encephalitis virus, and Huaiyangshan banyangvirus (formerly SFTS virus). Our results identified two I. ovatus ticks infected with Borrelia miyamotoi, which causes emerging relapsing fever; several I. nipponensis ticks infected with Rickettsia monacensis, which cause rickettsiosis; and several Ixodes persulcatus ticks infected with Rickettsia helvetica, which can also cause rickettsiosis. These results suggest that dogs and cats, and veterinary professionals and pet owners, in the Yamagata prefecture have some risk of exposure to several TBDs. This means that there should be continuous monitoring and reporting of TBDs, even those known to be uncommon in Japan, in both companion animals and humans to ensure the health and safety of both humans and animals in Japan.

Hemalin from Haemaphysalis flava ticks: cloning, expression and antithrombogenicity.

Hemalin, initially described in Haemaphysalis longicornis, is a protein with anticoagulant activity. We retrieved a gene fragment functionally annotated as hemalin from H. flava salivary gland transcriptomic library, but its full-length complementary DNA (cDNA) and antithrombogenicity have not been investigated in the species. Here we cloned the full length of hemalin (Hf-hemalin) by 3'-end rapid-amplification of cDNA ends, and the open reading frame (ORF) of Hf-hemalin was expressed in Escherichia coli. The recombinant protein (rHf-Hemalin) was tested for antithrombogenicity. The full-length of Hf-hemalin was 607 bp with an ORF of423 bp. Protein encoded by Hf-hemalin was predicted to contain 2 Kunitz domains and a signal peptide. The expression of Hf-hemalin in salivary glands, midguts and ovaries was higher in the semi-engorged than the fully engorged. Prokaryotic expression yielded a product of 40 kDa containing a glutathione S-transferase (GST) tag. Incubation of rHf-Hemalin with rat plasma significantly extended prothrombin time and activated partial thromboplastin time compared with normal saline and GST controls. Our data demonstrated that Hemalin from H. flava shared a similar primary structure with that from H. longicornis, and was also anticoagulant. Further investigations are needed to test its feasibility to be an antigen candidate for the development of vaccines against ticks.

Molecular Detection of Toxoplasma Gondii in Haemaphysalis Ticks in Korea.

Toxoplasma gondii are intracellular protozoa that can cause neurological disease or death in fetuses and even in immunocompromised human adults. Ticks are recognized as vectors of many microorganisms including viruses, bacteria, and protozoa. Recent studies detected T. gondii in various tick species in many countries. In this study, we performed PCR detection of the T. gondii B1 gene from Haemaphysalis ticks collected from vegetation in 4 localities, Wonju, Gunsan, Miryang, and Yangsan, in Korea. We analyzed DNA from 314 ticks (268 Haemaphysalis longicornis and 46 Haemaphysalis flava) and the B1 gene of T. gondii was detected in 13 of these. The detection of T. gondii in ticks differed significantly by region (P=0.021). T. gondii was detected in the following percentages of collected ticks: 3.7% (7 of 189) in Gunsan, 10% (5 of 50) in Wonju, 16.7% (1 of 6) in Yangsan, and 0% (0 of 69) in Miryang. The detection of T. gondii in ticks was not associated with tick species or development stage. This is the first report of T. gondii detection in ticks in Korea. Our results provide important information necessary to understand toxoplasmosis transmission.

The mitochondrial genome of the giant panda tick Haemaphysalis flava (Acari, Ixodidae) from Southwest China

The tick Haemaphysalis flava (Acari, Ixodidae) is an obligatory blood-feeding ectoparasite of the giant panda and is also a vector for transmission of pathogenic microorganisms. In this study, the complete mitochondrial genome of this tick was sequenced through Illumina sequencing technology. The genome was 14,699 bp in length and encoded 37 genes including 13 protein-coding genes, 22 transfer RNAs and two ribosomal RNAs. Phylogeny revealed that three isolates of H. flava, regardless of host origins and locations, clustered together and formed a monophyletic relationship with Haemaphysalis japonica, supporting their species validity among the genus Haemaphysalis. These cumulative mitochondrial DNA data provides insights into phylogenetic studies among Haemaphysalis ticks.

Molecular and Phylogenetic Analysis of Tick-Borne Pathogens in Ticks Parasitizing Native Korean Goats (Capra hircus coreanae) in South Korea.

Tick-borne pathogens (TBPs) are considered zoonotic re-emerging pathogens, with ticks playing important roles in their transmission and ecology. Previous studies in South Korea have examined TBPs residing in ticks; however, there is no phylogenetic information on TBPs in ticks parasitizing native Korean goat (NKG; Capra hircus coreanae). The present study assessed the prevalence, risk factors, and co-infectivity of TBPs in ticks parasitizing NKGs. In total, 107 hard ticks, including Haemaphysalis longicornis, Ixodes nipponensis, and Haemaphysalis flava, were obtained from NKGs in South Korea between 2016 and 2019. In 40 tested tick pools, genes for four TBPs, namely Coxiella-like endosymbiont (CLE, 5.0%), Candidatus Rickettsia longicornii (45.0%), Anaplasma bovis (2.5%), and Theileria luwenshuni (5.0%) were detected. Ehrlichia, Bartonella spp., and severe fever with thrombocytopenia syndrome virus were not detected. To our knowledge, this is the first study to report CLE and T. luwenshuni in H. flava ticks in South Korea. Considering the high prevalence of Candidatus R. longicornii in ticks parasitizing NKGs, there is a possibility of its transmission from ticks to animals and humans. NKG ticks might be maintenance hosts for TBPs, and we recommend evaluation of the potential public health threat posed by TBP-infected ticks.

A survey of proteins in midgut contents of the tick, Haemaphysalis flava, by proteome and transcriptome analysis.

Tick blood meals are stored and digested in their midguts. Blood digestion is complex, and many proteins are involved. Study of the tick-derived proteins in the midgut contentmay aid in the discovery of active molecules that would be useful for anti-tick vaccines. We analyzed the midgut content proteomes of partially engorged female Haemaphysalis flava, fully engorged female H. flava, and hedgehog serum using liquid chromatography tandem-mass spectrometry and label-free quantitation. In this study, high-confidence protein profiling of tick midgut content was determined. Based on the search against our in-house transcriptome database, the 28 high-confidence proteins were identified. Of these, 17 were identified as tick-derived, and the rest were of unspecified origin (proteins that could not be differentiated as host-derived or tick-derived proteins). The function of these midgut content proteins identified here may involve nutrient transportation, anti-coagulation, erythrocyte lysis, detoxification, lipid metabolism, and immunization. The presence of hemoglobin suggested that the red blood cells were lysed in the gut lumen. The midgut contents contain a large amount of fibrinogen and it has the ability to clot immediately. The midgut contained mostly host-derived proteins,andthese host proteins provide richnutrients for tick development and reproduction. However, some intracellular proteins were also identified, suggesting the possibility of shedding of the midgut epithelium and ingestion of saliva during feeding. This finding advances our understanding of the digestive mechanism and will be useful in the screening of vaccine antigens.

Cloning of four HSPA multigene family members in Haemaphysalis flava ticks.

The heat shock protein 70 (HSPA) family and their genes have been studied in ticks and are considered as possible antigen candidates for the development of anti-tick vaccines. However, knowledge about their members, structure and function in ticks is incomplete. Based on our transcriptomic data, the full length of four HSPA genes in Haemaphysalis flava (Acari: Ixodidae) was cloned via rapid amplification of cDNA ends. The open reading frame of HSPA2A, HSPA2B, HSPA5 and HSPA9 was 1920, 1911, 1983 and 2088 bp in length, respectively. Three family signatures and one localization motif were in the encoding proteins. HSPA2A and HSPA2B were predicted to be located at cytoplasm/nucleus, whereas HSPA5 and HSPA9 were at endoplasmic reticulum and mitochondria, respectively. In silico simulation demonstrated that those proteins had distinct numbers of α-helixes, extended strands and coils, and different antigenic epitopes. Expression of HSPA5 and HSPA9 in the salivary gland was significantly higher in partially-engorged female adult ticks than the fully-engorged (P < 0.01) as shown by a quantitative polymerase chain reaction. Our data indicated that H. flava ticks had at least four HSPA genes encoding proteins with different cellular locations, structures and expression profiles, suggesting their diverse roles in tick biology.

Detection of Borrelia DNA in Tick Species Collected from Vegetation and Wild Animals in Fukuoka, Japan.

We screened for the presence of Borrelia spp. in ticks collected from vegetation by flagging and from wild animals between May 2017 and November 2018 in Fukuoka, located in the northern Kyushu region of Japan. A total of 1,601 ticks were collected and separated based on morphology into nine species, namely Ixodes turdus, I. ovatus, Amblyomma testudinarium, Haemaphysalis flava, H. formosensis, H. kitaokai, H. longicornis, H. hystricis, and H. megaspinosa. The ticks were segregated into 561 pools and nested PCR was used to detect borrelial DNA. Borrelia turdi and Borrelia sp. HM were identified in two of the 561 pools. This is the first report of the presence of the Lyme disease group of Borrelia and of the relapsing fever group of Borrelia in Fukuoka, Japan.