Occurrence of Borrelia and Borreliella species in Ixodes ricinus collected from roe deer in northwestern Spain

Ixodes ricinus, comprising the predominant tick species in Europe, can transmit important human pathogens, including Borreliella spp., the causal agent of Lyme borreliosis. One hundred and seventy five roe deer hunted in two areas (plateau and mountain) of Galicia (northwest Spain) were examined for the presence of ticks; all roe deer were infested by I. ricinus. Nymphs (n = 1000), males (n = 1449) and females (n = 1000) of I. ricinus were analysed in pools of up to 10 ticks to detect both Borreliella and Borrelia DNA. The average number of I. ricinus per roe deer was similar in both areas, regardless of the life stage; although the percentage of Borreliella and Borrelia positive pools was higher in ticks collected from roe deer hunted in the plateau area, no significant differences were detected. Sequence analysis at the flagellin gene allowed the identification of four Borreliella species (Borreliella afzelii, Borreliella garinii, Borreliella lusitaniae and Borreliella valaisiana) and Borrelia miyamotoi in adult males; only B. valaisiana and B. miyamotoi were detected in nymphs and all females were negative. All Borreliella and Borrelia species found in roe deer were previously identified in questing I. ricinus collected in the same study area, although the prevalence was lower in the present study. The analysis of male I. ricinus ticks collected from roe deer gives a good estimation of Borreliella diversity in questing ticks.     

The relationships between Ixodes ricinus and small mammal species at the woodland–pasture interface

Ixodes ricinus, as vector, and small mammals, as reservoirs, are implicated in pathogen transmission between wild fauna, domestic animals and humans at the woodland–pasture interface. The ecological relationship between ticks and small mammals was monitored in 2005 on four bocage (enclosed pastureland) sites in central France, where questing ticks were collected by dragging and small mammals were trapped. Questing I. ricinus tick and small mammal locations in the environment were assessed through correspondence analysis. I. ricinus larval burden on small mammals was modeled using a negative binomial law. The correspondence analyses underlined three landscape features: grassland, hedgerow, and woodland. Seven small mammal species were trapped, while questing ticks were all I. ricinus, with the highest abundance in woodland and the lowest in pasture. The small mammals were overall more abundant in hedgerow, less present in woodland and sparse in grassland. They carried mainly I. ricinus, and secondarily I. acuminatus and I. trianguliceps. The most likely profile for a tick-infested small mammal corresponded to a male wood mouse (Apodemus sylvaticus) in woodland or hedgerow during a dry day. A. sylvaticus, which was the only species captured in grassland, but was also present in hedgerow and woodland, may be a primary means of transfer of I. ricinus larvae from woodland to pasture.     

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Woodland biodiversity management as a tool for reducing human exposure to Ixodes ricinus ticks: A preliminary study in an English woodland

This paper presents preliminary findings towards developing a UK‐specific approach to reducing public exposure to woodland questing Ixodes ricinus tick populations by harnessing existing biodiversity‐enhancing woodland ride (i.e., linear non‐wooded herbaceous habitat either side of track within woodland) management strategies. This preliminary study in an English woodland firstly assesses whether ecological and environmental factors determine presence and density of questing Ixodes ricinus along woodland rides. Secondly, it sets these findings in the context of woodland ride management guidelines in England in order to understand what impact ride management strategies might have on numbers of questing ticks and tick survival. Nymph and adult I. ricinus presence and abundance were modelled in relation to relevant microclimate and ecological parameter variables. Predictor variables for increased questing nymph abundance included ride orientation, mat depth, occurrence of bracken/bramble and animal tracks, ride/path width, and sward height. Ticks thrive in the ecotonal habitat of a woodland ride, therefore we urge woodland managers to consider the impact of their ride management on ticks and human exposure to ticks. Possible recommendations for mitigating questing I. ricinus in line with biodiversity management guidelines rides are discussed in this paper and include seasonal mowing regimes, management of mulch/mat, and bracken/bramble management through use of scalloped ride edges.     

Models of temporal variation in questing activity in individuals of Ixodes recinus (Acari: Ixodidae)

Intensive observations of the questing activity of Ixodes ricinus ticks in the field were made to provide data on the range of durations of periods of continuous questing activity, and of the variation in questing activity between individuals. Continuous periods of questing were observed to extend to a maximum of 28 hours. Substantial variation in questing activity between individuals was observed. Models fitted to the distribution of durations of bouts of questing activity provide insights into the questing ecology of I. ricinus. Results suggest that questing duration may not be solely dependent on the state of hydration of the tick. A function fitted to the frequency distribution of the proportions of active life that individuals spend on questing, provides an empirically-based model that can be used to generate a stochastic expression of variation of questing activity in individuals in a questing population.     

Boophilus microplus Infection by Beauveria amorpha andBeauveria bassiana: SEM Analysis and Regulation of Subtilisin-like Proteases and Chitinases

Beauveria bassiana is a well-known broad-range arthropod pathogen which has been used in biological control of several pest insects and ticks such as Boophilus microplus. Beauveria amorpha has both endophytic and entomopathogenic characteristics, but its capacity for biological control has still not been studied. During the processes of host infection, B. bassiana and B. amorpha produce several hydrolytic extracellular enzymes, including proteases and chitinases, which probably degrade the host cuticle and are suggested to be pathogenicity determinants. To access the role of these enzymes during infection in the tick B. microplus, we analyzed their secretion during fungus growth in single and combined carbon sources, compared to complex substrates such as chitin and B. microplus cuticle. Chitin and tick cuticle-induced chitinase in both fungus and protease was induced only by tick cuticle. SEM analysis of B. amorpha and B. bassiana infecting B. microplus showed apressorium formation during penetration on cattle tick cuticle.     

The importance of passerine birds as tick hosts and in the transmission of Borrelia burgdorferi,the agent of Lyme disease: a case study from Scotland

Borrelia burgdorferi sensu lato (s.l.) is the causative agent of Lyme borreliosis, the most common tick‐borne zoonosis of humans in Europe and North America. Here, we assessed the relative importance of different passerine bird species as tick hosts and their contribution to the B. burgdorferi s.l. transmission cycle in a rural residential area in Scotland. We caught 1229 birds of 22 species during the tick‐questing season. On average, 29% carried larval ticks (0.8 larvae per individual) and 5% carried nymph ticks (0.06 nymphs per individual). All attached ticks tested were Ixodes ricinus. Using a nested‐PCR, we found that 20% of nymphs tested positive to B. burgdorferi s.l. and all these were of the genospecies Borrelia garinii. We identified two new bird species carrying infected nymphs: Eurasian Siskin Carduelis spinus and European Greenfinch Carduelis chloris. Ground‐foraging species were more important than arboreal species in hosting I. ricinus nymphs and B. burgdorferi s.l. Common Blackbirds Turdus merula were the most common hosts, with Song Thrushes Turdus philomelos, Dunnocks Prunella modularis, European Greenfinches and Chaffinches Fringilla coelebs also hosting high rates of infection.     

Being a parasitoid of parasites: host finding in the tick wasp Ixodiphagus hookeri by odours from mammals

The parasitic wasp Ixodiphagus hookeri Howard (Hymenoptera: Encyrtidae) parasitizes larvae and nymphs of a number of tick species worldwide. Ticks themselves are parasitic on vertebrate hosts. To study the specificity and reliability of vertebrate odours used by I. hookeri for host location, we conducted bioassays in a four‐chamber olfactometer. Wasps were arrested by carbon dioxide and by odours from roe deer faeces and odours from hair of roe deer and wild boar. Odours from faeces of cattle, rabbit, and field mouse as well as odours from hair of cattle and field mouse had no effect. Odours from faeces of the host tick species Ixodes ricinus L. (Acari: Ixodidae) were attractive only up to a distance of 1 cm. Thus, I. hookeri reacts to general (carbon dioxide) and specific vertebrate odours from wild boar and deer. Examination of freshly shot specimens demonstrates that deer and wild boars are infested with a sufficient number of tick nymphs to tap the full reproductive potential of an I. hookeri female, which makes cues from these mammal species reliable. These results indicate that I. hookeri locates its hosts using specific and reliable mammal odours and that ticks are parasitized on their vertebrate hosts. The implications of this host‐finding strategy and its benefits for the parasitoid are discussed.     

Ticks in the ecotone: the impact of agri-environment field margins on the presence and intensity of Ixodes ricinus ticks (Acari: Ixodidae) in farmland in southern England

The present study aimed to assess whether agri-environment field margins provide a habitat for the sheep/deer tick Ixodes ricinus. Field studies were conducted in arable farmland in southern England in both extant and newly constructed field margins. The presence and intensity (i.e. the mean number of nymphs per transect, excluding zeros) of questing nymphs and adult I. ricinus were compared between field margins with three adjacent habitats: woodland, hedgerow and arable land. The presence and intensity of ticks within a field margin was also compared between three ecozones: the ecotone, the margin and the crop. It was found that field margins do support I. ricinus, although the intensity of ticks was associated with field margins with adjacent woodland, with a higher tick intensity along the ecotonal ecozone, compared with the rest of the margin or the crop edge. The presence of a hedge also increased the likelihood of finding questing nymphs in a field margin compared with a margin adjacent to arable land. This effect, however, was less pronounced than in field margins with adjacent woodland. The provision of footpaths within the margin (at least 1–2 m from the ecotone), or on the edge of the crop where paths run next to woodland known to be an important tick habitat, could be promoted to minimize tick exposure. In addition, based on the results of the present study, raising awareness that walking alongside woodlands also constitutes a tick risk could be promoted.     

Monitoring the diel activity of Ixodes ricinus ticks in Hungary over three seasons

Ixodes ricinus is the most common tick species in Europe and vector of many diseases of humans. The risk of contracting such a disease is influenced by many factors, but one of the crucial points is questing activity of unfed ticks. In order to supplement the few literature data on patterns of diel activity of this tick species and to examine the correlations between data on diel activity of ticks and their small mammal hosts and some meteorological variables, a survey was performed. Diel activity of questing I. ricinus and small rodents was studied in a known natural tick-borne encephalitis virus focus over 7 months at one sampling day monthly. 1,063 I. ricinus ticks and 25 rodents were sampled. Air temperature and humidity data were also recorded in the 24 study plots at time of sampling. From April to October questing activity of nymphs increased in the 3-h-period after sunrise, comparing to activity of the 3 h before sunrise. Proportion of nymphs sampled 3 h after sunset compared to total sampled nymphs 3 h before and 3 h after sunset showed correlation to activity of rodents. In the period of April–July both nymphs and larvae showed stronger activity from sunrise to sunset, this turned to dominant nighttime activity in August–September, whereas activity changed to equal in day and night in October. Our results indicate that natural light and rodent population have positive effect on questing activity of I. ricinus.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

Influence of selected meteorological variables on the questing activity of Ixodes ricinus ticks in Lower Silesia,SW Poland

The relationship between climate data and tick questing activity is crucial for estimation of the spatial and temporal distribution of the risk of ticks and tick‐borne diseases. This study establishes correlations between selected meteorological variables provided by the Weather Research and Forecasting model (WRF) and the questing activity of Ixodes ricinus nymphs and adults on a regional scale across Lower Silesia, Poland. Application of Generalized Linear Mixed Models (GLMM), built separately for adults and nymphs, showed that solar radiation, air temperature, and saturation deficit appeared to be the meteorological variables of prime importance, whereas the wind speed was less important. However, the effect of meteorological parameters was different for adults and nymphs. The adults are also more influenced by forest cover and the percentage of forest type if compared to nymphs. The WRF model providing meteorological variables separately for each location and day of tick sampling can be useful in studies of questing activity of ticks on a regional scale.     

Behavioural responses of Ixodes ricinus nymphs to carbon dioxide and rodent odour

Many haematophagous ectoparasites use carbon dioxide (CO₂) and host odour to detect and locate their hosts. The tick Ixodes ricinus (Linnaeus) (Ixodida: Ixodidae) walks only small distances and quests in vegetation until it encounters a host. The differential effects of CO₂ and host odour on the host‐finding behaviour of I. ricinus have, however, never been clarified and hence represent the subject of this study. The effects of CO₂ and odour from bank voles on the activation and attraction of I. ricinus nymphs were analysed in a Y‐tube olfactometer. Carbon dioxide evoked a response in the absence and presence of host odour, but did not attract nymphs. Host odour, however, did not evoke a response but did attract nymphs in the absence and presence of CO₂. The current results show that CO₂ is an activator, but not an attractant, and that host odour is an attractant, but not an activator, of I. ricinus nymphs, and provide ecological insights into the host‐finding behaviour of I. ricinus.     

Interstadial and Infestation Level-dependent Variation in the Transmission Efficiency of Borrelia Burgdorferi from Mice to Ixodes Ricinus Ticks

The efficiency with which the spirochaete Borrelia burgdorferi sensu stricto was transmitted from laboratory mice to larval and nymphal Ixodes ricinus ticks was assessed, using the polymerase chain reaction. The transmission efficiency to nymphs was significantly greater than to larvae when both fed together on the same host. Increased tick infestation levels of mice were correlated with significantly greater engorgement weights and higher B. burgdorferi transmission coefficients from mice to nymphs. These observations indicate that both the feeding success of ticks and the transmission coefficients from host to tick may be influenced by the tick infestation level of an infected host. The infestation level and the relative numbers of each life stage of the tick are factors which should be considered in the design of transmission experiments.     

Species,developmental stage and infection with microbial pathogens of engorged ticks removed from dogs and questing ticks

Research into tick‐borne diseases implies vector sampling and the detection and identification of microbial pathogens. Ticks were collected simultaneously from dogs that had been exposed to tick bites and by flagging the ground in the area in which the dogs had been exposed. In total, 200 ticks were sampled, of which 104 came from dogs and 96 were collected by flagging. These ticks were subsequently examined for DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia spp. and Babesia canis. A mixed sample of adult ticks and nymphs of Ixodes ricinus (Ixodida: Ixodidae) and Haemaphysalis concinna (Ixodida: Ixodidae) was obtained by flagging. Female I. ricinus and adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks dominated the engorged ticks removed from dogs. Rickettsia spp. were detected in 17.0% of the examined ticks, A. phagocytophilum in 3.5%, B. canis in 1.5%, and B. burgdorferi s.l. in 16.0%. Ticks with multiple infections were found only among the flagging sample. The ticks removed from the dogs included 22 infected ticks, whereas the flagging sample included 44 infected ticks. The results showed that the method for collecting ticks influences the species composition of the sample and enables the detection of a different pattern of pathogens. Sampling strategies should be taken into consideration when interpreting studies on tick‐borne pathogens.     

First survey on hard ticks (Ixodidae) collected from humans in Romania: possible risks for tick-borne diseases

The importance of studies on the diversity of ticks attacking humans resides mostly in the relatively highly-specific tick-pathogen associations. Human tick bites are commonly reported worldwide but removal of ticks from patients is rarely followed by specific identification of the ticks, leaving to some degree of hazard the preventive treatment of possible associated diseases. A total number of 308 ticks were collected between April and June 2010 from 275 human patients who voluntarily presented to a hospital from Cluj-Napoca, Romania. The mean intensity of infection was 1.12 ± 0.46. Four species of ticks were identified Ixodes ricinus, Dermacentor marginatus, Haemaphysalis concinna and H. punctata. Ixodes ricinus was the most abundant species feeding on humans in the study area. A brief review of possible associated pathogen is provided.     

Improvement of field sampling methods for adult Karoo paralysis ticks,Ixodes rubicundus (Acari: Ixodidae), through addition of host odour

The responsiveness of Ixodes rubicundus ticks on questing substrates and the success of their attachment to non-living substrates were investigated. The purpose of this study was to relate responsiveness to conditions of temperature and humidity, to compare the efficacy of flagging and dragging methods to estimate the size of populations of adult I. rubicundus in the field, and to determine the possible influence of host odour on the efficacy of these methods. Responsiveness was tested under varying conditions of temperature and humidity under field conditions, and the same ticks were used to determine the duration of attachment to a flannel cloth, either impregnated with host odour from sheep wool or without it. Flagging and dragging methods were compared under laboratory conditions with cloths either treated with host odour or not. Within the range of ambient temperatures recorded during this study (7–25°C) most of the ticks (86%) were responsive. No obvious relationship between temperature, relative humidity and responsiveness of ticks was evident. Ticks remained on average 32.9 seconds (n=64) on cloth treated with host odour compared to 9.1 seconds (n=54) on untreated cloth. Flagging was 1.5–1.7 times as effective as dragging and treatment of the cloths with host odour increased the efficacy 2.4 (dragging) to 2.8 (flagging) times. The reactions of ticks to external stimuli should, therefore, be taken into account to maximize field sampling success.     

First report of Anaplasma phagocytophilum and its co-infections with Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari: Ixodidae) from Republic of Moldova

We examined 198 questing Ixodes ricinus ticks collected in Chisinau City, Republic of Moldova by PCR assays for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and co-infection of both pathogens, which were detected in 9%, 25.2% and 2.5% of tested ticks, respectively. B. burgdorferi s.l. genotyping revealed the presence of five genospecies with dominance of B. garinii. Our preliminary study provides evidence about occurrence of both pathogens in this populated area, which represent a potential health risk for inhabitants.     

Host and pathogen DNA identification in blood meals of nymphal Ixodes ricinus ticks from forest parks and rural forests of Poland

DNA analysis of blood meals from unfed nymphal Ixodes ricinus allows for the identification of tick host and tick-borne pathogens in the host species. The recognition of host species for tick larvae and the reservoirs of Borrelia, Rickettsia and Anaplasma species were simultaneously carried out by analysis of the blood meals of 880 questing nymphal I. ricinus ticks collected in forest parks of Szczecin city and rural forests in northwestern Poland that are endemic areas for Lyme borreliosis. The results obtained from the study indicate that I. ricinus larvae feed not only on small or medium animals but also on large animals and they (i.e. roe deer, red deer and wild boars) were the most prevalent in all study areas as the essential hosts for larvae of I. ricinus. The composition of medium and small vertebrates (carnivores, rodents, birds and lizards) provided a more diverse picture depending on study site. The reservoir species that contain the most pathogens are the European roe deer Capreolus capreolus, in which two species of Rickettsia and two species of Borrelia were identified, and Sus scrofa, in which one Rickettsia and three Borrelia species were identified. Rickettsia helvetica was the most common pathogen detected, and other included species were the B. burgdorferi s.l. group and B. miyamotoi related to relapsing fever group. Our results confirmed a general association of B. garinii with birds but also suggested that such associations may be less common in the transmission cycle in natural habitats than what was thought previously.