Molecular analysis of <Emphasis Type="Italic">Ixodes granulatus</Emphasis>, a possible vector tick for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in Taiwan

The genetic identity of Ixodes granulatus ticks was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2–99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.     

Tick infestation risk and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> s.l. infection-induced increase in host-finding efficacy of female <Emphasis Type="Italic">Ixodes ricinus</Emphasis> under natural conditions

An investigation of the risk of human tick infestation, together with the prevalence of Borrelia burgdorferi s.l. infection, was conducted in a sylvatic habitat in western Germany to provide data needed for future risk-benefit evaluations of acaricides used for clothing impregnation. Additionally, data were collected on behavioural changes in Borrelia burgdorferi s.l.-infected adult female I. ricinus ticks and the possible impact of such changes on host-finding efficacy. The risk of I. ricinus-infestation was determined by collecting from the protective clothing of volunteers and by dragging in known tick-infested sites in the Kühkopf Mountain area, Koblenz, Germany, from June through October 2006. The overall tick infestation rate per person per hour was 7.4 ± 5.5, with the following sex- and stage-specific differences: males 0.32 ± 0.37, females 1.1 ± 1.2, nymphs 3.6 ± 4.4, larvae 2.4 ± 3.5. Concurrent dragging revealed an average 19.4 ± 16.2 times higher infestation rate as well as a markedly lower infection rate with borreliae in adult I. ricinus ticks when compared to ticks collected from exposed human volunteers. Although the difference in infection rates was statistically significant (P < 0.023) only in adult female ticks, our data indicate that B. burgdorferi s.l. infection may increase host-finding efficacy in adult I. ricinus. The overall exposure risk was 1.0 B. burgdorferi s.l.-infected ticks per person per hour of exposure, or 0.25 ticks per 100 m walking distance in the study area.     

<Emphasis Type="Italic">Dermacentor marginatus</Emphasis> and <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks versus L929 and Vero cell lines in <Emphasis Type="Italic">Rickettsia slovaca</Emphasis> life cycle evaluated by quantitative real time PCR

Ticks transmit many different pathogens to animals, humans and their pets. Rickettsia slovaca, as a member of the spotted-fever-group rickettsiae is an agent of the human disease Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), which occurs from the Mediterranean to central Europe, transmitted by Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). In this study, quantitative real time PCR was used to characterize the growth of R. slovaca, strain B in static (mammalian L929 and Vero cells without replacement of growth medium) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. Curves of bacterial growth in static cultivations were modeled with exponential, stationary and death phases, whereas in dynamic systems the stationary phase was absent. The highest point of multiplication of R. slovaca was recorded on the 4th day post infection in both cell lines and the rickettsial DNA copy number in L929 and Vero cells at this point was 21 and 27 times greater than rickettsial DNA copy number of inoculum, respectively. In the dynamic system, the highest point of multiplication was on the 21th and 12th day after feeding of ticks and rickettsial DNA copy numbers were 7,482 and 865 times greater than the inoculum in D. marginatus and I. ricinus, respectively. Life cycle of R. slovaca in mammalian cell lines was shorter; supposedly, bacteria destroyed these cells and ticks, especially D. marginatus, were considered a more appropriate environment.     

Comparative population dynamics of a generalist (<Emphasis Type="Italic">Ixodes ricinus</Emphasis>) and specialist tick (<Emphasis Type="Italic">I. hexagonus</Emphasis>) species from European hedgehogs

Although the population dynamics of the tick Ixodes ricinus are relatively well studied, those of other Western European tick species are largely unknown. Moreover, there is very little information related to the interactions between I. ricinus and other ticks. Such knowledge, however, is of special interest in respect to the epidemiology of tick-borne pathogens such as Borrelia spp. We compared the dynamics of the generalist I. ricinus with the nest-dwelling hedgehog specialist, I. hexagonus. Both species were collected from hedgehogs from a naturally infested experimental population between 2006 and 2008. Ticks were collected once a month from March to October from each hedgehog counted and the life history stage and species determined. All hedgehogs harboured both tick species. Nymphs, females and males of I. ricinus showed clear bimodal seasonal distributions with peaks in spring and autumn, while larvae peaked only in summer. The density of I. hexagonus life stages was low during the whole investigation period and seasonal fluctuations of population density were much weaker compared to I. ricinus. Nymphs and larvae showed comparatively little change in population size and no consistent period of peak density. Females showed a single peak in summer and males were found only occasionally on hedgehogs. We suggest density-dependent mechanisms regulating the population density of the specialist I. hexagonus but not of the generalist I. ricinus.     

Associations Between Coinfection Prevalence of <Emphasis Type="Italic">Borrelia lusitaniae</Emphasis>, <Emphasis Type="Italic">Anaplasma</Emphasis> sp., and <Emphasis Type="Italic">Rickettsia</Emphasis> sp. in Hard Ticks Feeding on Reptile Hosts

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host–vector–pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host–vector–pathogen system—green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia–Anaplasma coinfection rates.     

Differential diagnosis of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> and <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>: nymphs and larvae

We developed a method for differential diagnosis of nymphs and larvae of sheep (Ixodes ricinus (L.)) and taiga (I. persulcatus Sch.) ticks (Parasitiformes: Ixodidae) which allows to identify live material in the field.     

Molecular detection of <Emphasis Type="Italic">Rickettsia</Emphasis>, <Emphasis Type="Italic">Coxiella</Emphasis> and <Emphasis Type="Italic">Rickettsiella</Emphasis> DNA in three native Australian tick species

Three Australian native animal species yielded 60 samples composed of three indigenous ticks. Hosts included twelve koalas, two echidnas and one wombat from Victoria, and ticks were of the species Ixodes tasmani (n = 42), Bothriocroton concolor (n = 8) and B. auruginans (n = 10), respectively. PCR screening and sequencing detected a species of Coxiella, sharing closest sequence identity to C. burnetii (>98%), in all B. auruginans, as well as a species of Rickettsia, matching closest to R. massiliae, in 70% of the same samples. A genotype sharing closest similarity to Rickettsia bellii (>99%) was identified in three female B. concolor collected from one of the echidnas. Three samples of I. tasmani, taken from three koalas, yielded different genotypes of Rickettsiella. These results represent the first detection of the three genera in each tick species and identify a high level of previously undetected bacterial diversity in Australian ticks.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Influence of Abiotic Factors on Different Developmental Stages of the Taiga Tick <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> and the Sheep Tick <Emphasis Type="Italic">Ixodes ricinus</Emphasis>

This paper presents an analytical literature review of the effects of abiotic factors on the different developmental stages of the ixodid ticks Ixodes persulcatus and I. ricinus. These widespread species are generally well adapted to the potential variation gradient of the factors most important for their development, such as temperature and humidity, and also other environmental conditions. Their variation usually does not significantly affect the mortality of ticks at various “passive” stages of their development cycle. However, the number of generations of both species can vary if these factors influence the percentage of fully engorged diapausing individuals.     

PCR detection of re-emerging tick-borne pathogen, <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>, in deer ked (<Emphasis Type="Italic">Lipoptena cervi</Emphasis>) a blood-sucking ectoparasite of cervids

Anaplasma phagocytophilum is an obligate intracellular bacterium, circulating in the natural foci in enzootic, vector-host cycle. In Europe, A. phagocytophilum is transmitted by Ixodes ricinus ticks. In Slovakia, cervids which are considered as naturally infected reservoirs of A. phagocytophilum are besides the ticks commonly infested with insects from the family Hippoboscidae. In this study, the presence of A. phagocytophilum was confirmed in deer keds (Lipoptena cervi) removed from deer by using of molecular approach. Detection of A. phagocytophilum in deer keds represents the remains of infected blood meal taken from infected deer host, what underlines the potential role of these blood-sucking insects in the mechanical transmission of pathogenic bacteria within the susceptible population of wild animals. Moreover, it may suggest the risk for the transmission of A. phagocytophilum or related pathogens to humans and healthy animals via the bite of infected hematophagous ectoparasites.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

<Emphasis Type="Italic">Coxiella</Emphasis> Symbionts in the Cayenne Tick <Emphasis Type="Italic">Amblyomma cajennense</Emphasis>

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.     

Invasion of the Lyme Disease Vector <Emphasis Type="Italic">Ixodes scapularis</Emphasis>: Implications for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> Endemicity

Lyme disease risk is increasing in the United States due in part to the spread of blacklegged ticks Ixodes scapularis, the principal vector of the spirochetal pathogen Borrelia burgdorferi. A 5-year study was undertaken to investigate hypothesized coinvasion of I. scapularis and B. burgdorferi in Lower Michigan. We tracked the spatial and temporal dynamics of the tick and spirochete using mammal, bird, and vegetation drag sampling at eight field sites along coastal and inland transects originating in a zone of recent I. scapularis establishment. We document northward invasion of these ticks along Michigan’s west coast during the study period; this pattern was most evident in ticks removed from rodents. B. burgdorferi infection prevalences in I. scapularis sampled from vegetation in the invasion zone were 9.3% and 36.6% in nymphs and adults, respectively, with the majority of infection (95.1%) found at the most endemic site. There was no evidence of I. scapularis invasion along the inland transect; however, low-prevalence B. burgdorferi infection was detected in other tick species and in wildlife at inland sites, and at northern coastal sites in years before the arrival of I. scapularis. These infections suggest that cryptic B. burgdorferi transmission by other vector-competent tick species is occurring in the absence of I. scapularis. Other Borrelia spirochetes, including those that group with B. miyamotoi and B. andersonii, were present at a low prevalence within invading ticks and local wildlife. Reports of Lyme disease have increased significantly in the invasion zone in recent years. This rapid blacklegged tick invasion—measurable within 5 years—in combination with cryptic pathogen maintenance suggests a complex ecology of Lyme disease emergence in which wildlife sentinels can provide an early warning of disease emergence.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and Iberian red deer (<Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis>) in central Spain

Emerging tick-borne diseases of humans and animals have occurred frequently during the past 30 years. These disease outbreaks appear to result from changes in the distribution of tick and vertebrate hosts, and the introduction of humans and domestic animals into tick–pathogen–wildlife cycles. Use of molecular technologies now available for identification of pathogens in ticks can provide valuable information that allows for risk analysis of emerging tick-borne diseases. In this study, the prevalence of selected pathogens in ticks collected in six locations in central Spain from the major wild ungulate species, European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus), was determined by PCR. Tick species collected included Ixodes ricinus, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma m. marginatum. Pathogens identified in ticks included piroplasmids, Anaplasma spp., Ehrlichia spp. and Rickettsia spp. Piroplasmids were identified in all tick species except I. ricinus. Ehrlichia spp. were detected in all tick species and collection locations, while Rickettsia spp., which proved to be R. slovaca and a recently identified Rickettsia sp. DnS28, were identified only in D. marginatus. A. marginale and A. phagocytophilum were detected in D. marginatus, R. bursa and Hy. m. marginatum. Concurrent infections of these pathogens were frequently observed in ticks. Notably, A. phagocytophilum, which is infective for a broad host range that includes humans and domestic and wild animals, was identified in ticks from all collection locations. The variety of ticks and tick-borne pathogens demonstrated in this study suggests a risk in central Spain for the emergence of tick-borne diseases in humans and domestic animals.     

<Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> and other tick-borne bacteria in wild animals in western Slovakia

Tick-borne bacterial zoonoses of livestock and free-ranging ungulates caused by Anaplasma spp. are common in Central Europe. The aim of this study was to analyze the prevalence of Anaplasma phagocytophilum and other tick-borne bacteria in wild animals from western Slovakia. Infection with A. phagocytophilum was recorded in 62.86% of analyzed roe deer (Capreolus capreolus), in two red deer (Cervus elaphus) and two wild boars (Sus scrofa). Dermacentor reticulatus and Ixodes ricinus ticks collected on red deer were not A. phagocytophilum-infected. However, spotted fever group rickettsiae were detected in ticks collected from red deer. High prevalence of A. phagocytophilum in roe deer as well as positive red deer and wild boars suggest the occurrence of natural foci in western Slovakia.     

Detection of <Emphasis Type="Italic">Borrelia miyamotoi</Emphasis> in <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

Unfed adult ticks Ixodes persulcatus from five regions of Russia were examined by PCR method in order to analyze distribution and diversity of B. miyamotoi. B. miyamotoi DNA was found in 1.8, 2.9, 4.5, 2.3, and 2.5% of ticks from Leningrad, Sverdlovsk, Novosibirsk, and Irkutsk provinces, and from Khabarovsk Territory, respectively. Molecular typing of B. miyamotoi DNA was based on the partial sequencing of the 16S rRNA, p66, and glpQ genes. A single genetic variant of B. miyamotoi was detected in all the samples of ticks collected from five regions.