Ticks collected from selected mammalian hosts surveyed in the Republic of Korea during 2008-2009

A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%), I. nipponensis (1.8%), and H. japonica (0.2%).     

Phylogenetic analysis of spotted fever group rickettsiae based on gltA, 17-kDa, and rOmpA genes amplified by nested PCR from ticks in Japan

In order to understand the natural situation of rickettsiae in the ticks in Japan, the rickettsial genes, gltA gene, rOmpA gene, and 17-kDa gene, were amplified from the ticks by nested PCR. The prevalences of rickettsial gltA genes among Haemaphysalis formosensis, H. longicornis, H. megaspinosa, Ixodes ovatus, H. flava, H. kitaokai, and I. persulcatus were 62, 57, 24, 24, 19, 13, and 10%, respectively; 26% (186/722) being the average. The gltA genes amplified from the ticks were classified into 9 genotypes (I to IX) by the difference in nucleotide sequences. Genotype I was detected from 7 species of ticks. Genotype II mainly was detected from H. longicornis and H. formosensis. Genotypes III and VII mainly were detected from H. flava and I. ovatus. The polarization in the distribution of genotypes among regions where the ticks were collected was not clear. Based on the phylogenetic analysis of the three genes presented here, genotypes I, III, and IV (detected from H. formosensis, H. hystricia, and I. ovatus ) are genetically close with each other, but rickettsiae of the same property still have not been isolated from ticks anywhere in the world. These genotypes should be considered as new species among SFG rickettsiae. Genotype II was identical with strain FUJ-98, genetically close to R. japonica which has been isolated from ticks in China. Genotype V was identical with R. felis and strain California 2 isolated from the cat flea. This is the first report on the detection of R. felis from ticks. Genotype VI detected from Ixodes sp. did not seem to belong to genus Rickettsia. Based on the previous antigenic data and the phylogenetic analysis presented here, Genotype VII should be considered a variant of R. helvetica and genotype VIII detected from I. ovatus and I. persulcatus were identical with R. helvetica. Genotype IX detected from I. nipponensis was genetically close to the strains IRS3, IRS4, and IrR/Munich isolated from I. ricinus in Slovakia and German.     

Prevalence of Lyme disease Borrelia spp. in ticks from migratory birds on the Japanese mainland

Borrelia sp. prevalence in ticks on migratory birds was surveyed in central Japan. In autumn, a total of 1,733 birds representing 40 species were examined for ticks. A total of 361 ticks were obtained from 173 birds of 15 species, and these ticks were immature Haemaphysalis flava (94.4%), Haemaphysalis longicornis, Ixodes columnae, Ixodes persulcatus, Ixodes turdus, and an unidentified Ixodes species. Of these, 27 juveniles of H. flava on Turdus pallidus, Turdus cardis, or Emberiza spodocephala, 2 juveniles of I. persulcatus on T. pallidus, and 1 female H. flava molted from a T. pallidus-derived nymph were positive for the presence of Borrelia by Barbour-Stoenner-Kelly culture passages. In spring, a total of 16 ticks obtained from 102 birds of 21 species were negative for the spirochete. Isolates from 15 ticks were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis; all isolates were identified as Borrelia garinii with pattern B/B' based on the previous patterning. According to the intergenic spacer sequences, 2 of 15 isolates, strains Fi14f and Fi24f, were highly similar to B. garinii strains 935T of Korea and ChY13p of Inner Mongolia, China, respectively. These findings indicate that Lyme disease-causing B. garinii may have been introduced to Japan by migratory birds from northeastern China via Korea. Additionally, a case of transstadial transmission of B. garinii from nymph to adult H. flava suggests that the infected H. flava may transmit Borrelia to large animals.     

Interspecific and intraspecific associations of two species of hard ticks, Haemaphysalis longicornis and Haemaphysalis megaspinosa, in relation to questing site

The effects of intraspecific and interspecific interactions on preferred questing sites of ticks, specifically nymphs and larvae of Haemaphysalis longicornis and Haemaphysalis megaspinosa, were examined in Boso Peninsula of central Japan from October 1996 to September 1999. Haemaphysalis longicornis were primarily segregated from H. megaspinosa by season. All stages of the 2 tick species preferred sedges. Three-way contingency tables and log-linear models were used to test for independence of occurrence and to quantify associations between species and stages with similar host ranges. The shifts of questing site from leaves to stem tips and from 40-49 cm to greater heights were observed in both species, which suggests that these sites are more suitable for ticks and that aggregation may serve as protection from severe conditions. In contrast, a shift to a lower height was observed in H. longicornis nymphs and larvae when other species were present, suggesting that they were driven away by other species, especially H. megaspinosa. Heterospecific clusters composed of at least 2 species were formed on stem tips more frequently. It is concluded that questing site was affected by both aggregation pattern and the presence of other species.     

Survey of the vectorial competence of ticks in an endemic area of spotted fever group rickettsioses in Fukui Prefecture, Japan

The prevalence of SFGR in ixodid ticks in the Mt. Arashima-dake area in the northern part of Fukui Prefecture was surveyed, because of strong suspicions that the first case identified in this Prefecture had become infected with R. helvetica in this region. The ticks identified consisted of three genera and six species; I.ovatus, I. persulcatus, I. monospinosus, H. flava, H. japonica and D. taiwanensis. Of all 222 ticks collected, only I. monospinosus ticks (8 of 32 examined) were positive for SFGR isolates, which were genetically identified as R. helvetica. Ticks (157 of all 222) positive for SFGR-DNA fragments consisted of I. monospinosus (14 of 32), I. persulcatus (11 of 55), I. ovatus (3 of 38), H. flava (5 of 21) and H. japonica (2 of 9). Of these, thirteen I. monospinosus, eight I. persulcatus, three I. ovatus, two H. flava and one H. japonica were identified by nucleotide sequences as positive for R. helvetica. DNA fragments from three H. flava and one H. japonica showed greater homology to R. japonica than to R. helvetica or R. asiatica. The present results indicate that it is most likely that the vector tick of R. helvetica infection in Fukui Prefecture is I. monospinosus.     

Estimation of the density of nymphs of the bush tick, Haemaphysalis longicornis (Acari: Ixodidae), by the catch effort method

The density of nymphs of the bush tick, Haemaphysalis longicornis, was investigated by the catch effort method with flagging. The spatial distribution of H. longicornis nymphs fit the model of contagiously distributed colonies by Iwao's m*-m analysis (Iwao 1968). A sequential sampling method was used to predict the theoretical point at which to stop sampling. Our analysis showed that five quadrats (4 m x 4 m) were sufficient to estimate the density of H. longicornis nymphs with a mean density of 5.39 per quadrat. We estimated the tick density by two methods with respect to the sampling interval. The estimated density of ticks based on ticks collected during short sampling intervals (within a half hour) was 511.34 in the 18 quadrats with the extrapolation of the linear regression equation. On the other hand, for the long interval sampling, the total number of ticks estimated by the linear regression equation was 635.47 in six quadrats in which ticks had been collected by long interval sampling. There was a significant difference between the slopes of the two linear regression equations, suggesting that the rate of reduction in the number of H. longicornis nymphs in the study area by the catch effort method differed between the two sampling methods.     

Development, survival, fecundity and behaviour of Haemaphysalis (Kaiseriana) longicornis (Ixodidae) at two locations in southeast Queensland.

The free-living stages of the tick Haemaphysalis (Kaiseriana) longicornis were studied at Mt Tamborine (526 m altitude) and Amberley (25 m altitude) in southeast Queensland between 1971 and 1980. Data are presented on the number of eggs and larvae produced, the moulting success of engorged larvae and nymphs and the survival and behaviour of unfed larvae, nymphs and adult females. Temperature, moisture, daylength, grass length and age of unfed ticks were investigated as sources of variation in development rates, fecundity or survival. At Mt Tamborine the life cycle was well synchronized with the seasons to produce one generation per year. At Amberley higher temperatures accelerated development rates and would have delayed diapause, so disrupting the life cycle.     

Life cycle and host specificity of Amblyomma triste (Acari: Ixodidae) under laboratory conditions

We report biological data of two generations of Amblyomma triste in laboratory and compared the suitability of different host species. Infestations by larval and nymphal stages were performed on guinea pigs (Cavia porcellus), chickens (Gallus gallus), rats (Rattus norvegicus), rabbits (Oryctolagus cuniculus), wild mice (Calomys callosus), dogs (Canis familiaris) and capybaras (Hydrochaeris hydrochaeris). Infestations by adult ticks were performed on dogs, capybaras and rabbits. Tick developmental periods were observed in an incubator at 27 degrees C and RH 90%. Guinea pigs were the most suitable hosts for larvae and nymphs, followed by chickens. The remaining host species were less suitable for immature ticks as fewer engorged ticks were recovered from them. Mean larval feeding periods varied from 3.8 to 4.7 d between different host species. Mean larval premolt periods ranged from 8.9 to 10.4 d. Nymphal mean feeding periods varied from 4.2 to 6.2 d for ticks fed on different host species. Premolt period of male nymphs (mean: 15.4 d) was significantly longer than that of female nymphs (14.7 d). Female nymphs were significantly heavier than male nymphs. The overall sex ratio of the adult ticks emerged from nymphs was 0.9:1 (M:F). Capybaras were the most suitable host for the tick adult stage as significantly more engorged females were recovered from them and these females were significantly heavier than those recovered from dogs or rabbits. The life cycle of A. triste in laboratory could be completed in an average period of 155 d. The potential role of guinea pigs, birds and capybaras, as hosts for A. triste in nature, is discussed.     

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis.

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.     

Transmission of hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs, prevalent in Asia, Africa, and southern Europe. Experimental transmission of H. canis to dogs was performed with laboratory-reared Rhipicephalus sanguineus nymphs that fed on a naturally infected dog or were percutaneously injected with canine blood containing H. canis gamonts. Dogs were inoculated by oral ingestion of adult ticks containing H. canis oocysts. Transstadial transmission of H. canis was recorded, whereas transovarial transmission could not be demonstrated. Oocysts were detected in 85% of the adult ticks that had engorged as nymphs on an infected dog and in 61% of the adult ticks resulting from nymphs injected percutaneously with blood from the same dog. Nine of 12 dogs (75%) inoculated with naturally fed or percutaneously injected ticks became parasitologically positive, and all showed seroconversion. Meronts were initially detected in the bone marrow 13 days postinoculation and gamonts 28 days after infection. The variation in the time of initial detection of parasitemia among infected dogs and the rapid appearance of gamonts in dogs immunosuppressed with corticosteroids suggest that immune mechanisms play an important role in controlling H. canis parasitism. The artificial acquisition of Hepatozoon parasites by percutaneous injection of ticks, demonstrated here for the first time, may serve as a useful tool for studies on transmission, vector-host relationships, and the immunology of infection with Hepatozoon species.     

Tick infestation of dogs in Thessaloniki,northern Greece

Ticks were collected from 249 dogs, admitted over a one-year period (1996-1997) to a Veterinary Teaching Hospital and four private veterinary practices located in the county of Thessaloniki, Greece. A total of 2812 ticks (Acari: Ixodidae) were collected, of which 2511 (89.3%) were Rhipicephalus sanguineus (Latreille) adults (1070 males, 1441 females), 156 (5.5%) R. turanicus Pomerantsev adults (35 males, 121 females), 100 (3.6%) Rhipicephalus spp. nymphs and 45 (1.6%) Rhipicephalus spp. larvae. Dogs living outdoors, in rural areas and in close proximity to farm animals, were infested with higher numbers of ticks than dogs living indoors. Tick attachment sites were recorded on 237 dogs, harbouring a total of 1711 ticks. The most heavily infested sites were, in decreasing frequency, the ear pinnae, neck, interdigital skin folds, trunk, head, ventrum, extremities and the tail. Cutaneous lesions at the attachment sites were noticed in 129 dogs and then mainly in the more severely infested animals.     

Seasonal Distribution of Ticks in Four Habitats near the Demilitarized Zone,Gyeonggi-do (Province), Republic of Korea

This study describes the seasonal distribution of larvae, nymph, and adult life stages for 3 species of ixodid ticks collected by tick drag and sweep methods from various habitats in the Republic of Korea (ROK). Grasses less than 0.5 m in height, including herbaceous and crawling vegetation, and deciduous, conifer, and mixed forests with abundant leaf/needle litter were surveyed at United States (US) and ROK operated military training sites and privately owned lands near the demilitarized zone from April-October, 2004 and 2005. Haemaphysalis longicornis Neumann adults and nymphs were more frequently collected from April-August, while those of Haemaphysalis flava Neumann and Ixodes nipponensis Kitaoka and Saito were collected more frequently from April-July and again during October. H. longicornis was the most frequently collected tick in grass habitats (98.9%), while H. flava was more frequently collected in deciduous (60.2%) and conifer (57.4%) forest habitats. While more H. flava (54.1%) were collected in mixed forest habitats than H. longicornis (35.2%), the differences were not significant. I. nipponensis was more frequently collected from conifer (mean 8.8) compared to deciduous (3.2) and mixed (2.4) forests.     

Diversity and seasonal patterns of ticks parasitizing wild birds in western Portugal

The diversity and abundance of questing ticks and ticks parasitizing birds was assessed during 1?year in two recreational forests in western Portugal, a suburban forest and an enclosed game area. The aim of this study was to assess the distribution and seasonality of tick species and to understand the role of bird species as hosts for ticks. Ixodes ricinus was the most abundant questing tick collected in the enclosed game area, whereas in the suburban forest, only three ticks were collected by blanket dragging. Tick species parasitizing birds included I. ricinus, I. frontalis, I. arboricola, I. acuminatus, Haemaphysalis punctata, Hyalomma marginatum and H. lusitanicum. This is the first record of I. arboricola in Portugal. Tick prevalence and intensity of infestation differed between study areas and was higher in birds from the game area where a large population of deer and wild boar may support tick populations. Ground and shrub dwelling bird species such as Turdus merula, Erithacus rubecula and Sylvia melanocephala were the most heavily parasitized by ticks, but the importance of different bird species as hosts of larvae and nymphs of I. ricinus and I. frontalis differed. Therefore, different bird species may contribute differently for tick population maintenance.     

Regeneration of Haller's sensory organ in two species of hard ticks of the genus Haemaphysalis (Acari: Ixodidae)

A study of Haller's organ regeneration in nymphs and adults of Haemaphysalis turturis and parthenogenetic females of H. longicornis, from which the forelegs had been amputated during the previous instar, revealed structural changes in regenerated organs. The adult regenerates reestablished atavistic structural features, while the nymphal regenerates retained larval features, which is typical of regenerates of two other genera examined previously (Ixodes and Hyalomma). Data on regeneration of Haller's sensory organ testify to an ancient character of the genus, standing closely to the base of the phylogenetic tree of hard ticks.     

Spotted fever group Rickettsia in ticks from southeastern Spain natural parks

During an 8-years study, we collected from vegetation or domestic and wild mammals 1246 ticks (624 males, 511 females and 111 nymphs) belonging to 13 species in Jaen province (Andalusia) and we analyzed these ticks by PCR and sequencing for the presence of rickettsiae. Specific rickettsiae DNA was detected in 243 (19.5%) of the ticks tested. Sequence analysis of amplicons of gltA, ompA and ompB genes revealed that Ixodes ricinus were infected with R. monacensis, including strain IRS3, and R. helvetica (prevalences of 27.0% and 2.7%, respectively), while in I. ventalloi we found only this last species (12.5%). Moreover, Dermacentor marginatus presents R. slovaca (24.7%) and R. raoultii (59.9%). In Rhipicephalus sanguineus group ticks (Rh. sanguineus, Rh. turanicus and Rh. pusillus) only R. massiliae (15.2%) was found. Haemaphysalis punctata and Ha. sulcata were infected with a Rickettsia sp. near R. hoogstraalii (prevalence of 3.1% and 16.1%, respectively). In addition, Ha. punctata appeared infected with R. monacensis-like Rickettsia (1.0%) and R. raoultii (9.3%). None of I. hexagonus, Hyalomma lusitanicum, Hyalomma sp., Ha. hispanica or Rh. bursa studied ticks contained rickettsiae.     

Ectoparasites of the Pallas squirrel, Callosciurus erythraeus, introduced to Japan

The squirrel Callosciurus erythraeus (Pallas) (Rodentia: Sciuridae) was intentionally introduced to Japan in 1935 and has become established throughout much of the country. Although they live mainly in forests, Pallas squirrels come into gardens and are frequently fed by people or kept as pets, so their ectoparasites could be of potential medical as well as veterinary importance. During 2001-2003 we conducted the first ectoparasite survey of Pallas squirrels in Japan. From 105 C. erythraeus captured in Kamakura District of Kanagawa Prefecture on Honshu Island, three types of ectoparasite were found: 52 specimens of the sucking louse Neohaematopinus callosciuri Johnson (Anoplura: Haematopinidae), 26 fleas Ceratophyllus (Monopsyllus) anisus Rothschild (Siphonaptera: Ceratophyllidae) and four nymphs of the tick Haemaphysalis flava Neumann (Acari: Ixodidae) on 22, 13 and one squirrels, respectively. Evidently in Japan C. erythraeus carries relatively few ectoparasite species; this may be a contributory factor to their invasive success. Further investigations are needed to assess risks of zoonotic transmission of plague or murine typhus by C. anisus, of louse-borne typhus by N. callosciuri and of tularaemia and especially Japanese spotted fever (Rickettsia japonica) by H. flava.     

Tick species parasitizing people in an area endemic for tick-borne diseases in north-western Italy

In 1995 and 1996, 318 ticks were recovered from 240 people in Liguria (Province of Savona, Italy). Most of the ticks (284; 89.3%) were sheep ticks, Ixodes ricinus. Rhipicephalus sanguineus (31; 9.8%) and Dermacentor marginatus (3; 0.9%) were also recorded. All three life stages of I. ricinus were found on humans while only nymphs and adults of R. sanguineus and adults of D. marginatus were collected. Human tickbites were most frequent in the municipalities of the Province where roe deer density was highest. The number of tickbites in this area was about 500 per 100,000 residents over the period of observation. Most cases of tickbite were observed in children (11.2%), students (25.6%), workers (22.4%) and retired people (24%). Although ticks were recovered from people throughout the year, the highest frequencies of I. ricinus bites were in May, June and July. In the study area natural hosts were also studied. Six tick species were identified (I. ricinus, I. hexagonus, D. marginatus, R. bursa, R. sanguineus, R. turanicus). I. ricinus and R. sanguineus were the most abundant species. I. ricinus was recorded more frequently from ruminants particularly roe deer, while R. sanguineus was found to be associated with the dog.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

No Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Ixodid Ticks Collected in Seoul

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.     

The Use of a 4% (w/w) Deltamethrin Collar (Scalibor® ProtectorBand) in the Extended Control of Ticks on Dogs

Deltamethrin (4%, w/w) formulated in a polymer matrix system (Scalibor ProtectorBand, Intervet International BV) was evaluated for the prevention of ticks on dogs. Controlled laboratory trials were conducted, wherein dogs wearing anti-tick collars were compared with control dogs and artificially infested by adult Ixodes ricinus and/or Rhipicephalus sanguineus ticks. The activity of the collars increased from 24 to 48 h post-treatment (pt) wherein tick numbers were reduced to 78.7% for I. ricinus and to 77% for R. sanguineus. The proportion of ticks controlled after the second infestation on D7 pt increased to 99% for I. ricinus and 99.5% for R. sanguineus. For I. ricinus adult ticks, an efficacy of 95.4% was reached over a period of 5.5 months and approximated 90% for R. sanguineus. Hence, the efficacy of the deltamethrin-impregnated dog collar was >90% for 6 months against both tick species. There was no significant difference between the efficacy of the collar against either R. sanguineus or I. ricinus. A subsequent field study wherein 82 dogs were wearing the collar and monitored for tick infestation indicated that the duration of efficacy was between 5 and 6 months.