Members of the genus Borrelia are among the most common infectious agents causing tick-borne disease in humans worldwide. Here, we developed a Light Upon eXtension (LUX) real-time PCR assay that can detect and quantify Borrelia species in ticks that have fed on humans, and we applied the assay to 399 such ticks. Borrelia PCR-positive ticks were identified to species level by sequencing the products of conventional PCR performed using Borrelia group-specific primers. There was a 19% prevalence of Borrelia spp. in the detached ticks, and the number of spirochetes per Borrelia PCR-positive tick ranged from 2.0 ؋ 10 2 to 4.9 ؋ 10 5 , with a median of 7.8 ؋ 10 3 spirochetes. Adult ticks had a significantly larger number of spirochetes, with a median of 8.4 ؋ 10 4 compared to the median of nymphs of 4.4 ؋ 10 4 . Adult ticks also exhibited a higher prevalence of Borrelia (33%) than nymphs (14%). Among the identified species, Borrelia afzelii was found to predominate (61%) and was followed by B. garinii (23%), B. valaisiana (13%), B. burgdorferi sensu stricto (1%), B. lusitaniae (1%), and B. miyamotoi-like (1%). Also, 3% of the ticks were coinfected with multiple strains of B. afzelii. Notably, this is the first report of B. lusitaniae being detected in ticks in Sweden. Our LUX real-time PCR assay proved to be more sensitive than a corresponding TaqMan assay. In conclusion, the novel LUX real-time PCR method is a rapid and sensitive tool for detection and quantification of Borrelia spp. in ticks.Lyme borreliosis (LB) is the most common tick-borne disease in humans in Europe (26), and it is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex. That group comprises the species B. burgdorferi sensu stricto, B. afzelii, and B. garinii, which are usually transmitted by the vector Ixodes ricinus. Furthermore, there have been reports of B. valaisiana, B. lusitaniae, and B. spielmanii being detected in samples of human skin and cerebrospinal fluid (5, 7, 30), which suggests that those three species can also give rise to LB. It is often hard to distinguish the clinical symptoms of LB from those of other diseases (10), and hence, it can be difficult to establish a correct diagnosis, especially if the patient is unable to recall having a tick bite.Today, diagnosis is based mainly on serological tests, although some PCR-based approaches, such as the TaqMan real-time PCR assay (3, 12), have been developed to detect Borrelia species in clinical samples. Even if real-time PCR is not yet considered to be a routine method in clinical practice, it can nonetheless provide valuable information about Borrelia infections, with regard to species type and the number of spirochetes present. Additional major advantages of PCR in this context are its simplicity, sensitivity, robustness, and speed.Other assays besides the TaqMan assay include a method based on SYBR green dye chemistry (37) and another using Light Upon eXtension (LUX) (Invitrogen Corporation). Compared to the SYBR green real-time PCR assay, the LUX assay o...

The incidence of Lyme borreliosis (LB) in a region may reflect the prevalence of Borrelia in the tick population. Our aim was to investigate if regions with different LB incidences can be distinguished by studying the prevalence and diversity of Borrelia species in their respective tick populations. The Borrelia load in a feeding tick increases with the duration of feeding, which may facilitate a transmission of Borrelia Spirochetes from tick to host. Therefore, we also wanted to investigate how the Borrelia load in ticks that have fed on humans varies with the duration of tick feeding. During 2008 and 2009, ticks that had bitten humans were collected from four regions of Sweden and Finland, regions with expected differences in LB incidence. The duration of tick feeding was estimated and Borrelia were detected and quantified by a quantitative PCR assay followed by species determination. Out of the 2,154 Ixodes ricinus ticks analyzed, 26% were infected with Borrelia and seven species were identified. B. spielmanii was detected for the first time in the regions. The tick populations collected from the four regions exhibited only minor differences in both prevalence and diversity of Borrelia species, indicating that these variables alone cannot explain the regions’ different LB incidences. The number of Borrelia cells in the infected ticks ranged from fewer than ten to more than a million. We also found a lower number of Borrelia cells in adult female ticks that had fed for more than 36 hours, compared to the number of Borrelia cells found in adult female ticks that had fed for less than 36 hours.

BackgroundThe common tick Ixodes ricinus is the main vector in Europe of the tick-borne encephalitis virus and of several species of the Borrelia burgdorferi sensu lato complex, which are the etiological agents of Lyme borreliosis. The risk to contract bites of I. ricinus is dependent on many factors including the behaviour of both ticks and people. The tick’s site of attachment on the human body and the duration of tick attachment may be of clinical importance. Data on I. ricinus ticks, which were found attached to the skin of people, were analysed regarding potentially stage-specific differences in location of attachment sites, duration of tick attachment (= feeding duration), seasonal and geographical distribution of tick infestation in relation to age and gender of the tick-infested hosts.MethodsDuring 2008–2009, 1770 tick-bitten persons from Sweden and the Åland Islands removed 2110 I. ricinus ticks. Participants provided information about the date of tick detection and location on their body of each attached tick. Ticks were identified to species and developmental stage. The feeding duration of each nymph and adult female tick was microscopically estimated based on the scutal and the coxal index.ResultsIn 2008, participants were tick-bitten from mid-May to mid-October and in 2009 from early April to early November. The infestation pattern of the nymphs was bimodal whereas that of the adult female ticks was unimodal with a peak in late summer. Tick attachment site on the human body was associated with stage of the tick and gender of the human host. Site of attachment seemed to influence the duration of tick feeding. Overall, 63% of nymphs and adult female ticks were detected and removed more than 24 hours after attachment. Older persons, compared to younger ones, and men, compared to women, removed “their” ticks after a longer period of tick attachment.ConclusionsThe infestation behaviour of the different tick stages concerning where on the host’s body the ticks generally will attach and when such ticks generally will be detected and removed in relation to host age and gender, should be of value for the development of prophylactic methods against tick infestation and to provide relevant advice to people on how to avoid or reduce the risk of tick infestation.

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The prevalence of Candidatus Neoehrlichia mikurensis infection was determined in 102 persons bitten by ticks in Sweden. Two infected women had erythematous rashes; 1 was co-infected with a Borrelia sp., and the other showed seroconversion for Anaplasma phagocytophilum. Both patients had increased levels of Neoehrlichia DNA and serum cytokines for several months.