Dermacentor Silvarum Research Articles

Complete mitochondrial genomes of Dermacentor silvarum and comparative analyses with another hard tick Dermacentor nitens

Ticks are obligate blood-sucking ectoparasites of a wide range of vertebrates. They can transmit a range of pathogens that cause economic losses to livestock production as well as human disease. In the present study, the complete mitochondrial (mt) genome of Dermacentor silvarum was determined. The mt genome is 14,945 bp in length contains 37 genes, including 13 are protein-coding genes (cox1–3, nad1–6, nad4L, cytb, atp6 and atp8), two ribosomal RNA genes and 22 transfer RNA genes. The nucleotide composition of the D. silvarum mt genome was A + T biased at 78.78%; T was the most abundant nucleotide and G the least abundant. The mt genome of D. silvarum was 106 bp longer than that of Dermacentor nitens and the arrangements of two genomes were identical. For the 13 protein-coding genes, comparison between D. silvarum and D. nitens revealed sequence divergence at both the nucleotide (15.46–35.14%) and amino acid (6.05–48.98%) levels. Among them, cox1 was the most conserved gene, while atp8 was the least conserved. The lengths of the 13 protein-coding genes were the same or similar, except for cytb which was significantly longer in D. silvarum than in D. nitens. The mtDNA contained a variable repeat region consisting of a “similar to nad1” motif that was repeated three times, and the “Tick-box” motifs were also found. The overall difference between the nucleotide sequences of the two complete mt genomes was 21.4%. The mtDNA data presented in this study provide a rich resource for further studies on the phylogenetics, population genetics, and molecular epidemiology of ticks.

The first molecular evidence of severe fever with thrombocytopenia syndrome virus in ticks in Jilin, Northeastern China

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis. The aim of this study was to investigate SFTS virus (SFTSV) infections in ticks in Northeastern China. A total of 6427 ticks, including 5450 Haemaphysalis longicornis, 463 Dermacentor silvarum, 351 Dermacentor nuttalli, and 163 Ixodes persulcatus, were sampled in the Liaoning, Jilin, and Heilongjiang Provinces of Northeastern China. Viral megagenomic analysis of the ticks revealed 25 contigs targeting the M and L segments of the SFTSV genome. H. longicornis collected from Jinxing, Jilin Province, were analyzed by RT-PCR, which showed positive results for SFTSV, with a minimum prevalence of 3.0%. The full-length sequence of the S, M, and L segments of the SFTSV were obtained, and phylogenetic analysis showed that the virus strain found in Jilin formed a monophyletic cluster with the SFTSV strains from Jiangsu, suggesting that SFTSV in the Jilin Province may have spread from the Jiangsu Province. These findings are the first to demonstrate molecular evidence of SFTSV in ticks in the Jilin Province of Northeastern China and indicate the need for measures to prevent and control SFTS.

Genetic diversity and molecular characterization of Babesia motasi-like in small ruminants and ixodid ticks from China

Ovine babesioses, an important tick-borne disease of sheep and goats in China, is caused by the reproduction of intraerythrocytic protozoa of the Babesia genus. Babesia motasi-like is a Babesia parasite that infects small ruminant in China, and two sub-groups of B. motasi-like can be subdivided based on differences in the rhoptry-associated-protein-1 gene. This study aimed to characterize the distribution, epidemiology and genetics of B. motasi-like in animals and ticks. A molecular investigation was carried out from 2009 to 2015 in 16 provinces in China. In total, 1081 blood samples were collected from sheep and goats originating from 27 different regions, and 778 ixodid tick samples were collected from 8 regions; the samples were tested for the presence of B. motasi-like using a specific nested PCR assay based on the rap-1b gene. The results indicated that 139 (12.9%), 91 (8.4%), 48 (4.4%) and 6 (0.7%) of the blood samples were positive for general B. motasi-like, Babesia sp. BQ1 (Lintan and Ningxian), Babesia sp. Tianzhu and Babesia sp. Hebei sub-groups, mixed infections, respectively. Among the collected 778 ixodid ticks (including Haemaphysalis longicornis, Haemaphysalis qinghaiensis, Dermacentor silvarum, Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) microplus), the most frequently infected with Babesia were D. silvarum and I. persulcatus (35.7%), followed by H. longicornis (26.8%), H. qinghaiensis (24.8%) and R. sanguineus (9.3%). The PCR results were confirmed by DNA sequencing. The positive rates of B. motasi-like infection in ticks were found to be higher in China, compared with previous studies in other countries. B. motasi-like infections have not previously been reported in D. silvarum, I. persulcatus or R. sanguineus. The findings obtained in this study could be used for planning effective control strategies against babesiosis in China.

Species Determined Peculiarities of the Tick-Man Contacts in the South of the Primorsky Territory

Ticks’ ability to attack man (aggressiveness) is an important characteristic impacting the ticks’ epidemiological role. Objective of the study is to assess the aggressiveness of the ticks inhabiting the south of the Primorsky Territory. It is based on the materials collected in the territory of Partizansky Region in 2012-2013. Ticks’ aggressiveness toward man has been defined by comparing the species’ community structure when collecting imago specimens on a cloth from plants (3,944 specimens are examined) and when removing ticks from people (437). In total, five species are identified: Ixodes persulcatus , Haemaphysalis concinna , H. japonica douglasi, Dermacentor silvarum (being dominant species), and I. pavlovskyi (which is rarely found in Partizansky Region) . Cases of tick attacks have been registered during April through October and November: this period being the peak of activity in I. persulcatus , and activity decay in H. concinna. The percentage of I. persulcatus against the total number of ticks, removed from people, has amounted to 86.3 %; the percentage of the same species collected from plants has amounted to 44.9 %. This is maximum index of aggressiveness. I. pavlovskyi also shows high level of aggressiveness, but as it is of rare occurrence it does not have any epidemiological weight in Partizansky Region. H. japonica attacks more often than H. concinna. In order to assess the difference in the epidemiological significance of these two species individual infection rate should be studied. As for D. silvarum, the percentage of this species removed from people is lower than that collected from plants, thus currently the epidemiological significance of this species in the south of Primorsky Territory is relatively low.

Screening and Identification of Antigenic Proteins from the Hard Tick Dermacentor silvarum (Acari: Ixodidae).

In order to explore tick proteins as potential targets for further developing vaccine against ticks, the total proteins of unfed female Dermacentor silvarum were screened with anti-D. silvarum serum produced from rabbits. The results of western blot showed that 3 antigenic proteins of about 100, 68, and 52 kDa were detected by polyclonal antibodies, which means that they probably have immunogenicity. Then, unfed female tick proteins were separated by 12% SDS-PAGE, and target proteins (100, 68, and 52 kDa) were cut and analyzed by LC-MS/MS, respectively. The comparative results of peptide sequences showed that they might be vitellogenin (Vg), heat shock protein 60 (Hsp60), and fructose-1, 6-bisphosphate aldolase (FBA), respectively. These data will lay the foundation for the further validation of antigenic proteins to prevent infestation and diseases transmitted by D. silvarum.

Population dynamics of multiple symbionts in the hard tick, Dermacentor silvarum Olenev (Acari: Ixodidae)

Previously, we reported that Coxiella-like, Rickettsia-like and Arsenophonus-like symbionts could simultaneously coexist in Dermacentor silvarum. In this study, we examined their burdens and population dynamics in a single host during the host life cycle using quantitative PCR. Our results showed that multiple symbionts exhibited different abundances and varying trends in the tick host. Coxiella-like and Rickettsia-like symbionts were found at high densities in large quantities that fluctuated with time. This may coincide with oogenesis and mating of the host. Our findings provide insight into symbiont–tick interactions that lay the foundation for future studies.

The diversity and prevalence of hard ticks attacking human hosts in Eastern Siberia (Russian Federation) with first description of invasion of non-endemic tick species.

Hard ticks are the vectors of many pathogens including tick-borne encephalitis virus and the Lyme disease agent Borrelia burgdorferi sensu lato. In Eastern Siberia, Ixodes persulcatus, Dermacentor nuttalli, Dermacentor silvarum and Haemaphysalis concinna are regarded as aggressive to humans. Recently, significant changes in world tick fauna have been reported and this affects the spread of tick-borne pathogens. We studied the current species diversity, population structure and prevalence of tick-borne pathogens of hard ticks (Acari: Ixodidae) that attacked humans in Eastern Siberia (Irkutsk region, Russia). In total, 31,892 individual ticks were identified and analysed during the years 2007-2014. The majority (85.4%) of victims was bitten by I. persulcatus, 14.55% of attacks on humans were caused by D. nuttalli and D. silvarum, whereas H. concinna was documented only in 15 cases (0.05%). The seasonal activity and the age/gender structure of the tick population were studied as well. Among all the studied ticks, three unconventional species, i.e. Rhipicephalus sanguineus, Dermacentor reticulatus and Amblyomma americanum, were identified. Analysis of tick bite histories indicates at least three events of invasion of non-endemic ticks into the ecosystems of northern Eurasia with harsh continental climates. Invading ticks are able to reach the adult life stage and are aggressive to the local human population. Phylogenetic analysis of mt 16S rRNA gene fragments suggests multiple independent routes of tick migration to Eastern Siberia. Possible implications to human health and epidemiology of tick-borne infections are discussed.

Serologic Evidence for Babesia bigemina Infection in Wild Yak (Bos mutus) in Qinghai Province, China.

Piroplasmosis, caused by tick-borne protozoan parasites of the genera Theileria and Babesia, is one of the most economically important infections of domestic ruminants in China. We evaluated the prevalence of antibodies to Babesia bigemina in wild yaks (Bos mutus) from Qinghai Province, China. Of 91 samples, 24% had detectable B. bigemina antibodies, and significant differences in prevalence were observed between those older than 5 yr and those younger than 5 yr. We collected 1,679 ticks belonging to two Ixodid genera from the infested wild yaks. The most prevalent tick species was Dermacentor nuttalli (48.1%), followed by Haemaphysalis qinghaiensis (33.3%) and Dermacentor silvarum (18.6%). These results indicated that B. bigemina is most likely endemic in the study area, and active surveillance programs for piroplasmosis are needed for monitoring animal health status.

Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia

The Altai Republic is a highly endemic area as far as tick-borne encephalitis (TBE) is concerned. The aim of the research was to study the effect of altitude on the risk of tick-borne encephalitis infection in the Altai Republic. The paper analyzes the following data: the study of ixodid ticks collected from the vegetation in 116 sites at the 200–2383m elevation above sea level in 2012–2014, TBE virus prevalence of these vectors, tick-bite incidence rate, and TBE incidence rate of the population.Species identification of 4503 specimens has shown that the most common species are Dermacentor nuttalli (45.3%), Ixodes persulcatus (33.1%), Dermacentor silvarum (9.4%), Dermacentor reticulatus (8.9%), and Haemaphysalis concinna (5.0%).A total of 2997 adult ixodid ticks were studied for the presence of the TBE virus; 2163 samples were examined by ELISA, while 834 specimens were tested by PCR. The TBE virus prevalence of Dermacentor spp. ticks in both reactions was significantly higher than of Ixodes persulcatus ticks (p<0.001).The work shows that the altitude is an important factor in the development of the epidemiological situation of tick-borne encephalitis: the higher the elevation of the area above sea level, the smaller the range of vectors. There is also a change of a leading species: in middle altitude (800–1700m above sea level) the virus is transmitted by ticks of D. nuttalli along with I. persulcatus, and in high mountains (above 1700m above sea level) D. nuttalli becomes an absolute dominant species. However, these species of ticks are less effective vectors than I. persulcatus. With the increase of altitude the tick-bite incidence rate decreases (r=−0.78, p<0.05), and TBE incidence also reduces (r=−0.67, p<0.05).

Molecular characterization of a defensin gene from a hard tick, Dermacentor silvarum

BackgroundTicks are distributed worldwide and considered as vectors of many human diseases. Tick defensins, a family of antimicrobial peptides, form the first line of defense against pathogens.FindingsA defensin-like gene, named Ds-defensin, was identified from a cDNA library of the hard tick Dermacentor silvarum collected from northeast China. The full-length cDNA of Ds-defensin was 225 bp, encoding a 74 amino acid peptide. The nucleotide sequence of Ds-defensin shared 98.2% similarity to putative defensin from Dermacentor marginatus. RT-PCR results suggested that Ds-defensin was extensively expressed in tick salivary gland and midgut, with a higher expression level in midgut. Ds-defensin showed broad antimicrobial activity against various Gram-positive and Gram-negative bacteria, as well as the fungus Candida albicans.ConclusionsWe characterized a functional defensin from D. silvarum of China. Ds-defensin showed bactericidal activity against various Gram-positive and Gram-negative bacteria. Ds-defensin can be expected to be introduced to the medical field as a new molecule with antibacterial activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0625-0) contains supplementary material, which is available to authorized users.

Genetic variability of Babesia parasites in Haemaphysalis spp. and Ixodes persulcatus ticks in the Baikal region and Far East of Russia

To study Babesia diversity in Ixodid ticks in Russia, Ixodes persulcatus, Haemaphysalis japonica, Haemaphysalisconcinna, Dermacentor silvarum, and Dermacentor nuttalli ticks collected in the Far East and Baikal region were assayed for the presence of Babesia spp. using nested PCR. In total, Babesia DNA was detected in 30 of the 1125 (2.7%) I. persulcatus, 17 of the 573 (3.0%) H. concinna, and 12 of the 543 (2.2%) H. japonica but was undetectable in any of the 294 analyzed Dermacentor spp. Partial 18S rRNA gene sequences were determined for all of the positive samples. Among the positive ticks, nine I. persulcatus were infected by Babesia microti ‘US’-type, five I. persulcatus were infected by Babesia divergens-like parasites, and 11 I. persulcatus were infected by Babesia venatorum. For all three of these species, the determined 18S rRNA gene sequences were identical to those of the Babesia genetic variants found previously in I. persulcatus in Russia. In addition, five I. persulcatus from the Baikal region and all of the positive Haemaphysalis spp. ticks carried 13 different sequence variants of Babesia sensu stricto belonging to distinct phylogenetic clusters. Babesia spp. from 29 ticks of different species collected in distinct locations belonged to the cluster of cattle and ovine parasites (Babesia crassa, Babesiamajor, Babesiamotasi, Babesiabigemina, etc.). Babesia spp. from four H. japonica ticks in the Far East belonged to the cluster formed by parasites of carnivores. One more Babesia sequence variant detected in an I. persulcatus tick from the Baikal region belonged to the cluster formed by parasites of cattle and wild cervids (B. divergens, Babesiacapreoli, B. venatorum, Babesiaodocoilei, etc.).

Cloning and molecular analysis of voraxin-α gene of Rhipicephalus (Boophilus) microplus.

To identify suitable targets for development of cross-protective tick vaccine, in silico analysis was attempted and male tick derived molecule, voraxin-α was targeted. The voraxin-α homologue of Rhipicephalus (Boophilus) microplus was cloned, sequenced and analyzed employing standard methods. The deduced amino acids sequence analysis of the 419bp cloned voraxin-α gene of R. (B.) microplus indicated very high (94.6%) similarity with voraxin-α of the R. appendiculatus and moderate to low identity with Amblyomma hebraeum, Dermacentor silvarum and Haemaphysalis longicornis. The results suggest that recombinant voraxin-α might be a good candidate as cross-protective anti-tick vaccine.

Rickettsia raoultii, the predominant Rickettsia found in Dermacentor silvarum ticks in China–Russia border areas

Since the year 2000, clinical patterns resembling tick-borne rickettsioses have been noticed in China-Russia border areas. Epidemiological data regarding species of the aetiological agent, tick vector prevalence and distribution as well as incidence of human cases in the areas are still sparse to date. In order to identify Rickettsia species occurring in the areas, we investigated Dermacentor silvarum collected in the selected areas. Rickettsia raoultii was the predominant Rickettsia found in D. silvarum evident with ompA, ompB, gltA and 17 kDa protein genes. The Rickettsia prevalence in D. silvarum appeared to be 32.25 % with no sex difference. The results extend the common knowledge about the geographic distribution of R. raoultii and its candidate vector tick species, which suggest an emerged potential threat of human health in the areas.

Gene Cloning, Expression and Immunogenicity of the Protective Antigen Subolesin in Dermacentor silvarum

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

Pathogenicity of five species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) to the ixodid tick Dermacentor silvarum Olenev (Acari: Ixodidae)

We determined the mortality of adult tick Dermacentor silvarum Olenev after it was exposed to five entomopathogenic nematode species: Steinernema carpocapsae BJ (Sc BJ), S. glaseri NC32 (Sg NC32), S. ceratophorum D43 (Sc D43), S. feltiae Otio (Sf Otio) and Heterorhabditis bacteriophora E-6-7 (Hb E-6-7). All five species had lethal effects on adult ticks, and caused significant decreases in the amount of eggs laid by engorged female ticks (P < 0.05). Among them, Hb E-6-7 and Sc BJ were the most pathogenic to female adult ticks, with mortalities ranging from 70 to 90% (P < 0.05). Engorged females were more susceptible than unfed and partially fed females. The cumulative mortalities of engorged females exposed to Hb E-6-7 and Sc BJ for 14 days were 90%, with LT50 at 6.3 and 7.0 days, respectively. Dose-dependent effect was observed in the oviposition of the ticks after they were exposed to nematodes at concentrations of 1000–5000 IJs/dish. When the concentration was higher than 5000 IJs/dish, dose dependent effects disappeared (P > 0.05). All five nematode species were lethal to unfed male ticks, and there was significant difference in mortality when compared to the control group (P < 0.05); however, the pathogenicity varied among them.

Genetic variability of anaplasmataceae bacteria determined in Haemaphysalis spp. and Dermacentor sp. Ticks on the territory of the Russian Far East

In total, 484 Haemaphysalis japonica, 359 Haemaphysalis concinna, and 221 Dermacentor silvarum collected in Amur oblast and Khabarovsk krai of the Russian Far East were examined regarding the presence of Anaplasmataceae bacteria using nested PCR. All positive samples were characterized by analysis of the 16S rRNA gene and/or groESL operone nucleotide sequences. Forty-nine H. japonica and three H. concinna were shown to contain DNA of two new Ehrlichia genetic variants. On the basis of 16S rRNA gene and groESL operone nucleotide sequences analysis, these genetic variants were found to be most closely related to Ehrlichia spp. revealed in Haemaphysalis spp. ticks in Japan. Four H. concinna from Amur oblast were shown to contain DNA of a new Anaplasma bovis genetic variant, which corresponded to the A. bovis genetic variant revealed in a red gray-backed vole and a Siberian chipmunk from the Far East. Three H. concinna and nine D. silvarum contained DNA of atypical bacteria that cannot be attributed to any Anaplasmataceae genera based on the determined sequences of the 16S rRNA gene fragments. The revealed atypical bacteria significantly differed from each other and did not form a separate genetic group on the basis of 16S rRNA gene sequences.

Anaplasma phagocytophilum Infection in Domestic Animals in Ten Provinces/Cities of China

A nationwide epidemiologic investigation of domestic animal infections has been conducted in nine provinces and one city during 2007-2010. Serum samples from a total of 707 goats, 433 cattle, and 219 dogs were collected for detecting Anaplasma phagocytophilum IgG antibody by immunofluorescence assays and the average seroprevalences were 10.05% for dogs, 3.82% for goats, and 0.69% for cattle, respectively. A total of 472 goats, 201 cattle, 102 dog blood clots, and 1,580 ticks were collected for polymerase chain reaction (PCR) amplifying A. phagocytophilum 16S rRNA genes and the PCR-positive rates were 26.69% for goats, 23.38% for cattle, and 10.89% for dogs. Six species were identified and the average PCR-positive rates were 58.3% for Dermacentor silvarum, 43.9% for Haemaphysalis longicornis, 12.5% for Ixodes persulcatus, 7.5% (3 of 40) for Boophilus microplus, and 5.2% for Rhipicephalus sanguineus, respectively. The evidence in the study indicated the zoonotic Rickettsia is highly prevalent in China.

Investigation of Borrelia spp. in ticks (Acari: Ixodidae) at the border crossings between China and Russia in Heilongjiang Province, China

ObjectiveTo investigate the precise species of tick vector and the Borrelia spirochete pathogen at the Heilongjiang Province international border with Russia. MethodsIn this study, ticks were collected from 12 Heilongjiang border crossings (including grasslands, shrublands, forests, and plantantions) to determine the rate and species type of spirochete-infected ticks and the most prevalent spirochete genotypes. ResultsThe ticks represented three genera and four species of the Ixodidae family [Ixodes persulcatus, Dermacentor silvarum, Haemaphysalis concinna and Haemaphysalis japonica]. Ixodes persulcatus had the highest amount of Borrelia burgdorferi sensu lato infection of 25.6% and the most common species of Borrelia isolated from Ixodes persulcatus was Borrelia garinii, strain PD91. ConclusionsOur results suggest that Borrelia garinii PD91-infected Ixodes persulcatus may be the principal cause of Lyme disease in the border crossing areas of Heilongjiang Province.

First report on the occurrence of Rickettsia slovaca and Rickettsia raoultii in Dermacentor silvarum in China

BackgroundRickettsioses are among both the longest known and most recently recognized infectious diseases. Although new spotted fever group rickettsiae have been isolated in many parts of the world including China, Little is known about the epidemiology of Rickettsia pathogens in ticks from Xinjiang Autonomous Region of China.MethodsIn an attempt to assess the potential risk of rickettsial infection after exposure to ticks in Xinjiang Uygur Autonomous Region of China, a total of 200 Dermacentor silvarum ticks collected in Xinyuan district were screened by polymerase chain reaction based on the outer membrane protein A gene.Results22 of the 200 specimens (11%) were found to be positive by PCR. Phylogenetic analysis of OmpA sequences identified two rickettsial species, Rickettsia raoultii (4.5%) and Rickettsia slovaca (6.5%).ConclusionsThis study has reported the occurrence of Rickettsia raoultii and Rickettsia slovaca in Xinjiang Autonomous Region of China and suggests that Dermacentor silvarum could be involved in the transmission of rickettsial agents in China. Further studies on the characterization and culture of rickettsial species found in Dermacentor silvarum should be performed to further clarify this. Additionally, the screening of human specimens for rickettsial disease in this region will define the incidence of infection.

Anaplasma phagocytophilumInfection in Ticks, China–Russia Border

To the Editor: Anaplasma phagocytophilum, an emerging human pathogen of public health importance, is transmitted to humans most commonly by tick bites (1). The agent has been detected in various species of Ixodes ticks around the world (2) and in Dermacentor silvarum ticks in northeastern People’s Republic of China (3), where 3 A. phagocytophilum strains were isolated from wild and domestic animals (4). In the Asiatic region of Russia adjacent to China, A. phagocytophilum was identified in Ixodes persulcatus ticks, and A. bovis in Haemaphysalis concinna ticks (5). Human granulocytic anaplasmosis was reported in the southern area of the Russian Far East that borders China (6). The objectives of this study were to investigate the prevalence of A. phagocytophilum in ticks collected from the China–Russia border and to characterize the agent by molecular biology techniques.