the lowe alpine mountain marathon ( lamm ) is an annual long - distance mountain running event held in the scottish highlands .
teams of 2 runners each navigate mountainous terrain , carrying all their equipment for an overnight camp . to suit competitors with different levels of expertise and fitness ,
the event comprises 2 days of racing over several routes of varying lengths , typically 2040 km . in june 2014 ,
the lamm took place around wester ross on the west coast of mainland scotland ( http://www.lamm.co.uk/2014/lamm2014map.jpg ) ; the competition area covered 500 km .
racing on the first day was completed by 624 competitors , of whom 566 went on to complete racing on the second day . before the event
, the objectives and methods of our study were circulated to all competitors via a lamm website ( no longer available ) and were reiterated to competitors in person the night before the race . as competitors passed through the finish area on the first day ,
they were given a 1.5-ml tube , containing 70% ethanol and labeled  1  and were asked to place any ticks they found on their bodies into the tube and then put the tube into a bin provided at the overnight midway camp .
we also offered a tick - removal service , which proved popular and served to encourage wider participation .
as competitors passed through the finish area on the second day , they were given an addressed envelope containing a 1.5-ml tube containing 70% ethanol and labeled  2 .
 competitors were asked to check themselves either immediately or when they returned home , to place any ticks found in the tube as before , and then to put the envelope containing the tube in the mail . after we processed the samples and collated the data
, we posted a summary of survey results ( at http://www.lamm.co.uk/2014/lamm_lymeresults_2014.pdf ) , and the link was emailed to all competitors .
this email also asked any competitor in whom lyme borreliosis developed after the lamm to contact the study team . to assess if borreliae were an environmental hazard in the area and provide an indication of the scale of this hazard , we surveyed the competition area of the event for questing ticks by blanket dragging at 3 sites , specifically the day-1 assembly area ( rough pasture used for sheep grazing ) surveyed the night before the event , the overnight midway camp ( heather , long grass ) , and pine woodland in lochcarron , located 2 km from the assembly area ( both surveyed during event ) ( table ) . these efforts also served to increase awareness of the ongoing study throughout the event .
each site was surveyed for 30 minutes by repeatedly dragging a 1-m wool blanket over the vegetation .
after each drag , all ticks were removed from the blanket and placed in 70% ethanol .
we recorded the life stages of all ticks and examined them microscopically to identify their species .
a dna extract was prepared from each tick and then incorporated as template into a real - time pcr , originally described for the detection of b. burgdorferi s.l . but subsequently found to have a specificity that includes b. miyamotoi ( 8) . to delineate borrelia species / genospecies , we incorporated as template extracts that yielded a product in this reaction into a b. burgdorferi s.l.specific nested pcr ( 9 ) and a b. miyamotoi  specific real - time pcr ( 10 ) .
products of the first of these reactions were sequenced , and unambiguous sequence data were used to determine borrelia genospecies ( 9 ) .
extraction / cross - contamination controls were co - processed with ticks at a ratio of 1:4 .
each pcr incorporated positive and negative ( reagents only ) controls . pcrs were prepared in a dedicated laboratory in which pcr products were never handled .
the lowe alpine mountain marathon ( lamm ) is an annual long - distance mountain running event held in the scottish highlands .
teams of 2 runners each navigate mountainous terrain , carrying all their equipment for an overnight camp . to suit competitors with different levels of expertise and fitness ,
the event comprises 2 days of racing over several routes of varying lengths , typically 2040 km . in june 2014 ,
the lamm took place around wester ross on the west coast of mainland scotland ( http://www.lamm.co.uk/2014/lamm2014map.jpg ) ; the competition area covered 500 km .
racing on the first day was completed by 624 competitors , of whom 566 went on to complete racing on the second day . before the event
, the objectives and methods of our study were circulated to all competitors via a lamm website ( no longer available ) and were reiterated to competitors in person the night before the race . as competitors passed through the finish area on the first day ,
they were given a 1.5-ml tube , containing 70% ethanol and labeled  1  and were asked to place any ticks they found on their bodies into the tube and then put the tube into a bin provided at the overnight midway camp .
we also offered a tick - removal service , which proved popular and served to encourage wider participation .
as competitors passed through the finish area on the second day , they were given an addressed envelope containing a 1.5-ml tube containing 70% ethanol and labeled  2 .
 competitors were asked to check themselves either immediately or when they returned home , to place any ticks found in the tube as before , and then to put the envelope containing the tube in the mail . after we processed the samples and collated the data
, we posted a summary of survey results ( at http://www.lamm.co.uk/2014/lamm_lymeresults_2014.pdf ) , and the link was emailed to all competitors .
this email also asked any competitor in whom lyme borreliosis developed after the lamm to contact the study team . to assess if borreliae were an environmental hazard in the area and provide an indication of the scale of this hazard , we surveyed the competition area of the event for questing ticks by blanket dragging at 3 sites , specifically the day-1 assembly area ( rough pasture used for sheep grazing ) surveyed the night before the event , the overnight midway camp ( heather , long grass ) , and pine woodland in lochcarron , located 2 km from the assembly area ( both surveyed during event ) ( table ) . these efforts also served to increase awareness of the ongoing study throughout the event .
each site was surveyed for 30 minutes by repeatedly dragging a 1-m wool blanket over the vegetation .
after each drag , all ticks were removed from the blanket and placed in 70% ethanol .
we recorded the life stages of all ticks and examined them microscopically to identify their species .
a dna extract was prepared from each tick and then incorporated as template into a real - time pcr , originally described for the detection of b. burgdorferi s.l . but subsequently found to have a specificity that includes b. miyamotoi ( 8) . to delineate borrelia species / genospecies , we incorporated as template extracts that yielded a product in this reaction into a b. burgdorferi s.l.specific nested pcr ( 9 ) and a b. miyamotoi  specific real - time pcr ( 10 ) .
products of the first of these reactions were sequenced , and unambiguous sequence data were used to determine borrelia genospecies ( 9 ) .
extraction / cross - contamination controls were co - processed with ticks at a ratio of 1:4 .
the species of all ticks encountered in our study was i. ricinus . on day 1 of the lamm ,
217 ticks were removed and submitted by 53 competitors , and on day 2 ( or later by mail ) , 347 ticks were removed and submitted by 78 competitors .
it is not known how many competitors submitted ticks on both days , but on the basis of a recorded 624 competitors on day 1 and 566 on day 2 , we an estimate that a minimum of 8.5% of competitors on day 1 and 13.8% of competitors on day 2 were bitten by ticks .
significantly more competitors were bitten on day 2 than on day 1 (   8.47 , 1 df , p<0.01 ) .
a total of 44 ( 7.1% ) competitors on day 1 and 54 ( 9.6% ) competitors on day 2 were bitten by nymphs .
a total of 25 ( 4.0% ) competitors on day 1 and 50 ( 8.8% ) competitors on day 2 were bitten by larvae .
both nymphs and larvae were removed from the skin of 16 ( 2.6% ) competitors on day 1 and 26 ( 4.6% ) competitors on day 2 .
each submission contained 028 larvae and 09 nymphs , although most submissions contained either only 1 nymph or 1 larva .
infection with b. burgdorferi s.l . was found in 3 ( 0.7% ) of larvae and 19 ( 12.4% ) of 153 nymphs that had fed on competitors .
we were able to further characterize 16 of these ticks : 1 larva was infected with b. afzelii , 1 larva and 2 nymphs with b. burgdorferi sensu stricto , 7 nymphs with b. valaisiana , 2 nymphs with b. garinii , 1 nymph with b. miyamotoi ; and 2 nymphs had mixed infections ( 1 with b. valaisiana and b. garinii and the other with b. afzelii and b. garinii ) . infected larvae were removed from 2 competitors , and infected nymphs were removed from 16 competitors . in 4 instances , > 1 tick removed from the same competitor was infected .
a total of 10 infected ticks ( 3 larvae and 7 nymphs ) were removed from 9 ( 1.4% ) of the 624 day-1 competitors , whereas 12 infected nymphs were removed from 9 ( 1.6% ) of the 566 day-2 competitors .
no competitor reported development of lyme borreliosis or symptoms compatible with b. miyamotoi infection subsequent to the lamm .
a total of 283 questing nymphal ticks were collected from the competition area and tested for the presence of dna belonging to borrelia spp .
we were able to further characterize infections in 11 of these ticks , finding 7 to be infected with b. afzelii , 3 with b. garinii , and 1 with b. valaisiana .
the prevalence of infection among nymphs removed from humans did not differ significantly from that among questing ticks (   0.75 , 1 df , p = 0.39 ) .
the species of all ticks encountered in our study was i. ricinus . on day 1 of the lamm ,
217 ticks were removed and submitted by 53 competitors , and on day 2 ( or later by mail ) , 347 ticks were removed and submitted by 78 competitors .
it is not known how many competitors submitted ticks on both days , but on the basis of a recorded 624 competitors on day 1 and 566 on day 2 , we an estimate that a minimum of 8.5% of competitors on day 1 and 13.8% of competitors on day 2 were bitten by ticks .
significantly more competitors were bitten on day 2 than on day 1 (   8.47 , 1 df , p<0.01 ) .
a total of 44 ( 7.1% ) competitors on day 1 and 54 ( 9.6% ) competitors on day 2 were bitten by nymphs .
a total of 25 ( 4.0% ) competitors on day 1 and 50 ( 8.8% ) competitors on day 2 were bitten by larvae .
both nymphs and larvae were removed from the skin of 16 ( 2.6% ) competitors on day 1 and 26 ( 4.6% ) competitors on day 2 .
each submission contained 028 larvae and 09 nymphs , although most submissions contained either only 1 nymph or 1 larva .
infection with b. burgdorferi s.l . was found in 3 ( 0.7% ) of larvae and 19 ( 12.4% ) of 153 nymphs that had fed on competitors .
we were able to further characterize 16 of these ticks : 1 larva was infected with b. afzelii , 1 larva and 2 nymphs with b. burgdorferi sensu stricto , 7 nymphs with b. valaisiana , 2 nymphs with b. garinii , 1 nymph with b. miyamotoi ; and 2 nymphs had mixed infections ( 1 with b. valaisiana and b. garinii and the other with b. afzelii and b. garinii ) . infected larvae were removed from 2 competitors , and infected nymphs were removed from 16 competitors . in 4 instances , > 1 tick removed from the same competitor was infected .
a total of 10 infected ticks ( 3 larvae and 7 nymphs ) were removed from 9 ( 1.4% ) of the 624 day-1 competitors , whereas 12 infected nymphs were removed from 9 ( 1.6% ) of the 566 day-2 competitors .
no competitor reported development of lyme borreliosis or symptoms compatible with b. miyamotoi infection subsequent to the lamm .
a total of 283 questing nymphal ticks were collected from the competition area and tested for the presence of dna belonging to borrelia spp . the overall prevalence of b. burgdorferi s.l .
we were able to further characterize infections in 11 of these ticks , finding 7 to be infected with b. afzelii , 3 with b. garinii , and 1 with b. valaisiana .
the prevalence of infection among nymphs removed from humans did not differ significantly from that among questing ticks (   0.75 , 1 df , p = 0.39 ) .
the presence of ticks at the site of the 2014 lamm was entirely expected , as was presence of b. burgdorferi s.l .
infections in these ticks . in a recent survey of 25 sites across scotland , primarily in the highlands ,
infections in questing nymphs and in nymphs removed from competitors ( 48/436 , 11.0% ) and the diversity of b. burgdorferi s.l .
genospecies encountered was akin to that previously reported in scotland and elsewhere in the united kingdom ( 8,11 ) .
the presence of b. miyamotoi in scotland has not been reported previously , but a study of questing nymphs in england suggested that the pathogen is widespread , albeit at a very low prevalence ( 3/954 , 0.3% ) ( 12 ) .
perhaps the most unexpected observation was the high proportion of competitors bitten by ticks ; our estimates are that 8.5% were bitten on day 1 and 13.8% on day 2 .
of 131 competitors who reported being bitten by ticks , 33 ( 25% ) found larvae only .
dna in 0.7% of larvae from competitors supports the work of others ( 1315 ) , indicating that although rates of infection may be relatively low among larvae , ticks at this life stage should not be disregarded as a source of lyme borreliosis .
low prevalence of b. miyamotoi infections in i. ricinus larvae collected in mainland europe has been demonstrated ( 15 ) .
our failure to detect this species in any of the 411 larvae we tested indicates that , on the event site at least , infection of larvae is extremely rare .
the remaining 98 submissions , representing 75% of all those bitten by ticks , found either nymphs only or both nymphs and larvae .
nymphs have far greater potential to transmit b. burgdorferi s.l . ; in this study , prevalence of infection among nymphs was 18 times higher than that among larvae .
of 98 nymph submissions , 16 ( 16% ) were infected with either b. burgdorferi s.l . or b. miyamotoi ; 1.1% of day-1 competitors and 1.6% of day-2 competitors removed infected ticks from their bodies . over the 2 days of the lamm , competitors assembled at the camping area at 6 pm on the evening before the start and departed after finishing the race 2 days later
given that we encountered ticks not just on the lamm course but also in the camping area used by the competitors when not racing , we can reasonably assume that the only periods when competitors were not exposed to ticks was when they were inside their tents .
we therefore estimate that through the 48 hours of the event , 624 competitors were exposed to ticks for 32 hours , a total of 20,000 competitor - hours .
the total of 564 tick bites occurring during this period indicate 1 bite every 35 competitor - hours . of these ,
153 were from nymphs ; thus , 1 nymph tick bite occurred every 131 competitor - hours ; a bite from an infected nymph occurred once every 1,051 competitor - hours .  
these data provide an early quantification of the frequency of human exposure to b. burgdorferi s.l . in the vicinity of the lamm and ,
given the estimated abundance of nymphal ticks in this area and the prevalence of b. burgdorferi s.l .
infections among these ticks , may represent values encountered more widely across scotland and other parts of the united kingdom ( 8,11 ) .
extrapolation of these data may offer a tentative insight into frequency of human exposure in similar tick - infested habitat across the country .
however , as well as caveats associated with geographic / spatial variation in the environmental hazard posed by b. burgdorferi s.l .
, other determinants of tick exposure need also to be considered , including anthropogenic determinants .
for example , the degree to which competitors behavior was representative of countryside users at large ( or a specific cohort thereof ) is unknown ; the infrequency with which lamm competitors used clear footpaths may have resulted in their encountering questing ticks more often , and their light apparel ( many competitors wore shorts and short - sleeved tops ) may have enabled attachment of more ticks .
in addition , abiotic factors such as the influence of local weather conditions on tick questing behavior during the lamm and the topography of the course , sections of which were at relatively high altitudes where ticks are less abundant ( 16 ) , need to be considered . of note
, we observed more frequent tick bites on the second day of competition , despite persistent light rain throughout the morning , an observation in keeping with previous reports that ticks remain active in rain and that rainfall is associated with increased tick activity ( 17 ) .
however , we can not rule out that this observation resulted from competitors simply being more vigilant after the competition finished . various approaches to exploring the frequency of human exposure to ticks
these approaches vary from the quantification of ticks accumulated on fabricated leggings worn by investigators moving over / through tick - infested vegetation ( thus , rate of encounter rather than attachment ) ( 1,18 ) to assessment of the number of patients seeking help from medical staff for tick removal ; in 1 such survey of visitors to a popular woodland site in southern england during april 
october 1996 and 1997 , ticks were removed from almost 1,100 persons ( 19 ) .
however , only very few studies have attempted to quantify the rate at which users of tick - infested habitat actually get bitten .
one survey , of 568 soldiers at an outdoor training base in germany , recorded 710 tick bites during april 
september 2009 , a mean incidence rate of 2.3 bites/1,000 person - days ( 20 ) .
more recently , a survey of 931 scouts attending summer camps in belgium yielded a mean incidence rate of 22.8 tick bites/1,000 person - days ( 21 ) . both of these incidence rates are markedly lower than that reported in the current study , which equates to 677 bites/1,000 competitor - days .
why such variation exists is unclear ; it may be artifactual , resulting from differences in the efficiency of tick detection in 3 studies , or it may be genuine .
if the latter , it is worth considering that our survey was conducted in early june , when the abundance of immature ticks is probably near its seasonal peak ( 22 ) , whereas the surveys in germany and belgium extended over several months , during some of which tick abundance is probably reduced .
indeed , in the study in germany , > 95% of all tick bites occurred during 3 of the 6 months ( april 
also , in belgium , marked differences in incidence rates were observed between camps ; a range of 097.9 bites/1,000 person - days were recorded ( 21 ) .
none of the 3 surveys under discussion included accurate estimates of questing tick densities at the survey sites ; however , given that the questing tick densities at the 3 lamm sites surveyed were not unusually high ( mean of 189 ticks / person - hour , which equates to 98/100 m ( 23 ) , it is unlikely that the 100-fold variation in mean incidence rate of tick bites between our study and that of faulde et al .
( 20 ) is solely a reflection of variation in local tick abundance . perhaps more pertinent
is that the participants of the study in germany wore battle dress uniforms and were provided with arthropod - repellent skin cream , whereas the lamm competitors wore light apparel , frequently shorts and short - sleeved tops , thus making it easier for ticks to access their skin ( and possibly attracting ticks more by unimpeded release of attractant molecules from the skin )
21 ) did not provide details of what the participants in their study wore or the nature of their activities , but given that they were all scouts 816 years of age , it is reasonable to assume their clothing was more akin to that of lamm competitors than that of german soldiers . that none of the competitors reported clinical symptoms compatible with either b. burgdorferi s.l . or b. miyamotoi infections subsequent to the lamm is most likely the result of the well - established prophylactic benefits of removing ticks within a few hours of attachment
. however , it may also reflect a low transmission rate of b. burgdorferi s.l . from ticks to humans ( 19,24 ) or a low incidence of clinical manifestations among those infected ( 25,26 ) . in summary ,
our study demonstrates how mass - participation outdoor recreational events can be used to assess human exposure to dangers such as tickborne pathogens . by using one such event
, we were able to survey a large number of persons very efficiently ; we obtained more samples in just 2 days than previous studies with similar aims ( 19,20 ) obtained in 6 months .
mass - participation outdoor recreational events are now more diverse and popular than ever ; for example , thousands of orienteers and mountain / hill / trail runners participate in events every weekend , and vast numbers attend music or arts festivals throughout the summer .
furthermore , participants generally have a strong connection with the outdoors and so are probably willing contributors to scientific studies linked to nature .
a refinement to our approach would be to track more precisely the movement of persons through the landscape .
this tracking could be achieved by focusing on events at which participants follow a fixed course or wear gps ( global positioning system ) tracking devices ( as is becoming increasingly common ) , akin to a study quantifying the risk for tick infestation of dogs ( 27 ) .
 mapping the public health threat of tickborne pathogens requires quantification of not only the density of infected host - seeking ticks but also the rate of human exposure to these ticks . to efficiently sample a high number of persons in a short time , we used a mass - participation outdoor event . in june 2014 , we sampled 500 persons competing in a 2-day mountain marathon run across predominantly tick - infested habitat in scotland . from the number of tick bites recorded and prevalence of tick infection with borrelia burgdoferi sensu lato and b. miyamotoi 
 , we quantified the frequency of competitor exposure to the pathogens . 
 mass - participation outdoor events have the potential to serve as excellent windows for epidemiologic study of tickborne pathogens ; their concerted use should improve spatial and temporal mapping of human exposure to infected ticks . 
Materials and Methods
Sample Collection 
Identification of Ticks and Determination of Infection Status
Results
Survey 
None
Survey of the Environment
Discussion