lyme disease ( ld ) is a multisystem infectious bacterial disease caused by borrelia burgdorferi and is transmitted by the tick vector ixodes scapularis . this disease is considered the most prevalent arthropod - borne disease in the united states ( us ) . in recent years , there has been an increase in the number of human ld cases confirmed by the centers for disease control and prevention ( cdc , 2015 ) across its geographic distribution . in addition , recent studies project that ld is more prevalent than previously expected with over 300,000 infected individuals annually , and therefore significantly under - reported ( kuehn , 2013 ) . to date , most studies investigating ld prevalence in the us have focused on the endemic northeastern and midwestern states ( lane and burgdorfer , 1986 , gill et  al . , 1994 , ostfeld and keesing , 2000 , pepin et  al . , nevertheless , in recent years several studies on ld in the texas - mexico transboundary southern us region have emerged ( illoldi - rangel et  al . , 2012 , clark et  al . , 2014 , feria - arroyo et  al . , 2014 , , 2014 , szonyi et  al . , 2015 ; mitchell et  al . , in addition , a number of reports about the isolation of b.  burgdorferi spirochetes from humans , as well as from i.  scapularis ticks removed from animals in texas were published during the 1980a and 1990 s ( burgdorfer and keirans , 1983 , rawlings , 1987 , rawlings et  al . , 1987 , piesman and sinsky , 1988 , teltow et  al . b.  burgdorferi sensu stricto causes ld while b.  afzelii and b.  garinii are considered the cause of most european cases . other borrelia genospecies possibly associated with human clinical cases are b.  valaisiana , b.  bissettii , b.  americana , and the recently discovered b.  mayonii ( genospecies number 19 ) ( ryffel et  al . the ticks responsible for the transmission of this pathogen are i.  scapularis and i.  pacificus . other ixodes species known to participate in the enzootic cycle of this bacterial pathogen in the us are i.  dentatus , i.  affinis and i.  uriae ( olsen et  al . , 1995 , brownstein et  al . , 2003 ) . ixodes are three - host ticks with a lifecycle spanning two to four years during which they undergo four developmental stages including egg , larva , nymph and adult . the larval and nymphal stages of these ticks feed on a wide host range including small mammals such as the white - footed mouse ( peromyscus leucopus ; a natural reservoir of b.  burgdorferi ) , chipmunks and squirrels , birds , reptiles , and also larger animals such as white - tailed deer ( wtd ) ( frank et  al . adult stages of ixodes prefer to feed on large mammals , especially wtd , odocoileus virginianus . wtd not only facilitate mating by the adult stages of ixodes , but also serve as a source of blood meal for female ixodes egg production ( main et  al . , 1981 , wilson et  al . nevertheless , wtd is not considered a reservoir for b.  burgdorferi ( ostfeld et  al . , 2006 ) . it is also worthy to note that wtd do not show symptoms of diseases when infected female ixodes feed on them . on the other hand , due to their role in maintaining tick populations , studies in the endemic areas of northeastern and midwestern us have shown that wtd densities and ixodes abundance correlated positively with human cases of ld . a lower density of wtd and number of ticks in these areas is correlated with lower numbers of reported cases of ld in humans ( wilson et  al . , 1985 , kilpatrick et  al . , 2014 ) . however , some studies showed that elimination of wtd does not remove the risk of ld in an area ( ostfeld and keesing , 2000 ) due to the complexity of the ecology of this disease . in addition , data regarding the ecology of ld in nonendemic southern us is very limited . serological tests have revealed the presence of antibodies to b.  burgdorferi in various animal species . these include wtd as well as other wild mammals ( white - footed mouse , raccoon ) , and domestic animals ( dog , cat , horse , cattle ) ( main et  al . , 1981 , magnarelli et  al . , 1984 , brownstein et  al . , 2003 ) . the application of serologic surveillance in wtd has been used to establish geographic locations where b.  burgdorferi circulates ( magnarelli et  al . , 1984 , magnarelli et  al . , 1986 , lane and burgdorfer , 1986 , gill et  al . , 1994 , martinez et  al . , 1999 ) . with little being known about ld ecology in the southern us ( esteve - gassent et  al . , 2015 , szonyi et  al . , 2015 ) , the detection of i.  scapularis ticks infected with b.  burgdorferi in texas ( feria - arroyo et  al . , 2014 ) , and the growing population of wtd nationwide ( rawinski and square , 2008 , mcshea , 2012 , raizman et  al . , 2013 ) , the objective of the current study was to determine the sero - reactivity of texas wtd to b.  burgdorferi during a 15-year longitudinal study ( 20012015 ) . from october 2001 to february 2015 , a total of 1493 male and female wtd ranging from 0.5 to 6.5 years of age were sampled during the texas hunting season from 14 counties of the state . about 56.9% of the sampled wtd were adults ( two years and older ) , 23% were yearlings ( one to two years old ) , and 20.1% fawns ( less than one year of age ) . the counties from which samples were collected included bee , bell , brazos , gonzales , guadalupe , hamilton , karnes , kerr , medina , real , travis , uvalde , webb , and williamson ( fig .  1 ) . all blood samples were collected , centrifuged , sera separated and stored in a 20  c freezer until used . j. morrill from the university of texas medical branch ( utmb ) ( galveston , texas ) , and the orion research and management services , inc . alice blue - mclendon at the texas a&m university winnie carter wildlife center , served as negative controls . these were collected from 2003 to 2013 from pen - raised wtd with no known exposure to ticks or b.  burgdorferi . these animals received ivermectin injections ( for its acaricidal properties ) triple the recommended dose annually in the fall , and repeated every 1014 days . in addition , a second group of negative controls were obtained in 2015 from wtd on deer ranches that implemented tick control measures , and where ixodes is less prevalent . indirect elisa was used to detect antibodies to b.  burgdorferi in the sera of wtd following previously described protocols ( small et  al . , this modification used b.  burgdorferi b31 strain a3 grown in ( barbour - stoenner - kelly ii ) bsk - ii medium ( ph 7.6 ) , and supplemented with 1% inactivated rabbit serum , at 32  c and 1% co2 . elisa plates were blocked with 3% bovine serum albumin ( bsa ) to reduce nonspecific reactivity . the primary antibody dilution used was 1:200 ( wtd serum samples ) and 1:2000 was used for the secondary antibody dilution ( horseradish peroxidase - conjugated rabbit anti - deer immunoglobin g , rockland immunochemicals , inc . both primary and secondary dilutions were carried out in 0.1  m phosphate - buffered saline ( ph 7.4 ) with 0.1% tween 20 . the substrate used for the enzyme included both o - phenylene diamine dihydrochloride ( opd ) ( thermo fisher scientific , life technologies , carlsbad , ca , usa ) and hydrogen peroxide . samples were considered sero - reactive when the optical density 450  nm ( od ) values were three standard deviations ( sd ) above the mean for the negative controls ( od  =  0.662 ) . commercially developed standardized western immunoblot assays for the analyses of wtd sera are not available . therefore , the samples with a high sero - reactivity ( high optical density above the cut off value ) when compared to the negative controls , were tested further with a standardized western immunoblot assay . this assay was used to determine the specificity of the immune reaction to b.  burgdorferi specific antigens , and to rule out false positives . the standardized western immunoblot assay used in this study this modification used b.  burgdorferi pure cell lysates , which were separated in 12% sds - page gels following standardized electrophoresis protocols ( maruskova et  al . after borrelia proteins were separated , gels were transferred to nitrocellulose membranes ( ge healthcare ) using the rta transfer blot kit ( bio - rad laboratories , inc . the membranes were blocked using 1% nonfat skimmed milk in tris buffer saline ( tbs ) containing 0.2% tween 20 . primary antibody ( wtd serum samples ) was utilized at 1:1500 dilution and incubated overnight at 4  c , while secondary antibody ( peroxidase conjugated rabbit anti - deer igg , rockland immunochemicals , inc . , limerick , pa , usa ) dilution at 1:5000 was incubated for one hour at room temperature . all blots were visualized using chemiluminescence ( bio - rad chemiluminescence and colorimetric detection kit , bio - rad laboratories , inc . , hercules , ca , usa ) and imaged using a chemidoc  touch ( bio - rad laboratories , inc . , hercules , ca , usa ) . samples with five or more bands ( excluding cross - reactive bands that were also present on the immunoblots of the negative control samples ) were considered sero - reactive , in line with the ld diagnostic standards established by the cdc , while samples with four or less bands were considered negative . the sero - reactive samples were further analyzed using the b.  burgdorferi ( igg ) marblot strip system ( trinity biotech plc . , bray , ireland ) following manufacturer s recommendations with modifications to adapt the protocol to wtd . unbound sera were washed from the strip , and bound b.  burgdorferi specific antibodies were reacted with alkaline phosphatase conjugated anti - deer igg . the strips were then washed to remove the unbound igg , and the strips were eventually reacted with a precipitating color developing solution , which deposited a purple precipitate on antibody - reacted bands . bands were visualized , and scored for intensity relative to the 41  kda band of the weakly reactive control and recorded . the statistical analysis used to determine the cut - off value for the indirect elisa was stata 12.0 statistical software ( stata , statacorp lp , college station , texas 77845 usa ) . the cut - off value ( fig . serum samples of 1493 wtd were collected and evaluated for sero - reactivity against b.  burgdorferi . of these samples , the indirect elisa results were read at the optical density of 450  nm ( od ) and ranged from 0 to 1.395 for the test samples , and 00.662 for the negative control samples . the cut off value ( 0.803 ) used to detect the highly sero - reactive samples was calculated by adding the average of the negative controls to three times their standard deviation . therefore , test samples above 0.803 were considered sero - reactive and thereafter further analyzed with standardized western immunoblot assay ( fig .  2 ) . a total of 65/1384 ( 4.7% ) wtd had a high sero - reactivity to b.  burgdorferi by indirect elisa and 7/1384 ( 0.5% ) by standardized western immunoblot assay ( table  1 ) . control samples ( 109 ) were utilized to evaluate cross - reactivity of wtd negative serum to b.  burgdorferi antigens . in this experiment , we observed that a number of bands ( 28  kda , 32  kda , 35  kda , 70  kda ) appeared cross - reactive because they were also found in the negative control samples ( fig .  2 ) . consequently , those four bands were removed from analyses of the test samples , and not considered when marking the highly sero - reactive wtd samples ( table  2 ) . the samples marked as highly sero - reactive were those that had five or more reactive bands by the standardized western immunoblot assay ( fig . consequently , from the sero - reactive group , 10.8% ( 7 out of 65 ) of the highly sero - reactive samples by elisa were also sero - reactive by standardized western immunoblot assay , which represented approximately 0.5% ( 7 out of 1384 ) of the total samples studied . the gender distribution of the sero - reactive samples by standardized western immunoblot assay was as follows ; 42.9% ( 3/7 ) were males ( two fawns and a yearling ) while 57.1% ( 4/7 ) were adult females . there is no apparent age or sex trend observed with respect to the sero - reactive samples . in addition , the sero - reactive samples by standardized western immunoblot assay were further tested using the commercially available marblot western blot assay ( fig . this assay is designed for qualitative in  vitro detection of human immunoglobulin g ( igg ) antibody to individual proteins of b.  burgdorferi ( b31 ) in human serum ( http://www.trinitybiotech.com ) to confirm diagnostics of lyme disease ( cdc , 1995 ) . as shown in fig .  3 , four of the seven sero - reactive wtd samples also provided a sero - reactive result using the commercially available marblot strip system . the presence of bands on these marblot strips indicated that specific antibodies to individual b.  burgdorferi proteins were present in the wtd sera . in particular , the reactive bands correspond with borrelia antigens at 66  kda , 60  kda , 58  kda , 34  kda . the remaining two samples provided a very weak immunoreactivity to the different borrelia antigens presented in this test , and therefore we consider those two samples negative for the test . overall , the counties with the highest prevalence of sero - reactive samples were travis and williamson , located in central region of texas ( fig .  1 ) . interestingly , those sero - reactive wtd samples were detected in years 2002 ( 6 samples ) and 2009 ( 1 sample ) , which correlate with the years in which texas reported to cdc their highest numbers of human ld ( fig . in addition , when looking at the density of wtd in the counties with the sero - reactive samples , we observed that they are located in the eco - regions with the highest wtd densities in the state of texas . furthermore , those densities were above state average , with a continuous increase since 2007 ( fig . the current study was designed to evaluate the sero - reactivity of wtd to ld pathogen ( b.  burgdorferi ) in central and south texas , and we showed a sero - reactivity of only 4.7% ( 65/1384 ) by indirect elisa and 0.5% ( 7/1384 ) by standardized western immunoblot assay . in addition , the sero - reactive samples are distributed across different eco - regions of texas , in particular , the central region of the state ( texas parks and wildlife department , 2015 ; https://tpwd.texas.gov/education/hunter-education/online-course/wildlife-conservation/texas-ecoregions ) . additionally , the years of high wtd sero - reactivity correlate with the years during which there was a peak of reported ld cases in texas ( fig .  4 ) . the observed discrepancy between the elisa and western blot results presented in this study may be due to potential cross - reactivity of wtd igg with antigens from different borrelia species . the animals tested were in sylvatic environments and may have had multiple bites by different tick species that might have exposed them to a number of bacterial species with antigenic structures similar to those present in the b.  burgdorferi cultures used for the elisa assay . for instance , the presence of relapsing fever borrelia such as b.  turicatae and b.  lonestari has been documented in texas ( barbour et  al . , 1996 ; ( whitney et  al . , 2007 ) . these bacterial species have been shown to have cross - reactive antigens with b.  burgdorferi ( bunikis and barbour , 2002 ) . taking together , this could explain the higher number of sero - reactive animals by elisa . the negative control samples were obtained from pen - raised wtd at the texas a&m university winnie carter wildlife center , with no known exposure to ticks or b.  burgdorferi ; deer ranches where ectoparasites and acarid control measures were implemented ; and areas where there was a lower possibility of ixodes survival . due to the fact that wtd are free roaming animals and do not get spirochetemia when exposed to b.  burgdorferi infected ticks , the objective of our study was to obtain a large pool of negative samples , including those with known lack of tick exposure , as well as those that could have been exposed to ticks but not to the bacterial pathogen , to account for a more realistic background reactivity of wtd serum samples in the different serological tests performed . upon further analysis of the 65 samples that were sero - reactive by elisa with standardized western immunoblot assay , samples with five or more bands were considered highly sero - reactive , while samples with four or less bands were marked negative . there is a lack of standardized tests for wtd and any information on their reactivity to specific b.  burgdorferi antigens . therefore , the decision of high sero - reactivity was based on the ld diagnostic standards of the center for disease control and prevention ( cdc ) and the immunoblot results obtained in this study ( fig .  3 ) . some of the reactive bands seen with the samples sero - reactive by standardized western immunoblot assay were consistent with the bands seen on western immunoblots of samples from humans with ld ( craft et  al . , 1986 , grodzicki and steere , 1988 , gill et  al . , 1993 ) . in the current study , the 28  kda , 32  kda , 35  kda and 70  kda borrelia antigens may be nonspecific as these were found in most of the wtd serum samples negative by elisa , as well as the negative control samples ( fig . , only one of the seven samples sero - reactive by western immunoblot assay showed a response to one of the two major outer surface proteins , ospb ( 34  kda ) ( table  2 ) . however , animals immunized with killed ld spirochetes have been shown to respond to the outer surface proteins ospa and ospb ( gill et  al . , 1993 ) . previous studies have also reported a lack of response to the outer surface proteins of b.  burgdorferi in humans infected in the early stage of ld ( craft et  al . , 1986 , grodzicki and steere , 1988 , guy , 1993 ) . therefore , we could hypothesize that the sero - reactive animals had a recent exposure to b.  burgdorferi , because our sampling period ( fall and winter months in texas ) correlates with the season in which i.  scapularis adult ticks shows its highest activity . three of the seven sero - reactive wtd samples did not give a positive result when the marblot strip system was used , but these samples showed a number of reactive bands in the developed in - house immunoblot test , and high elisa readings . this discrepancy could be due to the fact that other borrelia species such as b.  lonestari , the causative agent of southern tick associated rash illness ( stari ) , is also present in the state of texas . in this respect , b.  lonestari is known to be transmitted by amblyomma americanum ( lone star tick ) , which also feeds on wtd , and co - infections could be present . unfortunately , the sero - reactivity of our wtd samples against b.  lonestari antigens was not possible to evaluate due to the lack of any available isolate . this is due to the fact that currently , there is no isolate of b.  burgdorferi from texas that may be used to immunize or infect wtd in order to generate a positive control group . moreover , the conditions that will make wtd seroconvert have not yet been clearly established . in addition , this is the first study in texas , so there is no prior data collection to compare our new wtd data . therefore , we evaluated the high sero - reactivity based on a comparison with our negative controls . taken together , it is therefore likely that , the generally low sero - reactivity recorded in this serological analysis of wtd in texas , may be an indication of a low incidence of ld in this non - endemic southern region of the us . in reviewing the literature , no data was found on the correlation of a high population density of wtd with ld incidence in texas , but there have been reports of a positive correlation between wtd population and ld cases in endemic regions of the us ( wilson et  al . , 1985 , kilpatrick et  al . , 2014 ) . nonetheless , there has been an increase in population density of wtd from 14,000 to 3.8 million across texas over the past decade ( alan , 2013 ) . as shown in fig .  5 , the highly sero - reactive wtd were reported in one of the texas eco - regions in which wtd population density has significantly increased ( alan , 2013 ) over the past decade . therefore , we hypothesize that the increased populations of wtd may be a contributing factor to the increased ld cases in these two counties . moreover , this study is unique by virtue of being the first of its kind with such large number of samples obtained over an extended 15-year longitudinal period ( 20012015 ) . the results from this study serve as the underlying basis for further exploration of the important factors that contribute to the pathogenic landscape of ld in texas . in addition , through the evaluation of wtd sera across multiple continuous years , we have provided experimental data that will facilitate the development of accurate ld ecology models in non - endemic regions of the country , rather than using extrapolations and/or assumptions from models generated in endemic areas . from our data , the overall seroprevalence for b.  burgdorferi in wtd in texas is low , but two counties in the central part of the state in which we recorded higher levels of antibodies correlate with the years during which a peak of human ld cases occurred . additionally , we have demonstrated , for the first time , the distribution of b.  burgdorferi antibodies in wtd in texas . future research is aimed at geographical information system ( gis ) mapping methods and spatio - temporal analyses to evaluate land use changes over the period of time during which these serum samples were obtained . this study will be relevant to understand the link between wtd sero - reactivity to b.  burgdorferi , land use changes in texas , the observed increase in wtd populations in the central region of the state , and their impact on ld risk in non - endemic areas .
lyme disease is caused by the bacterial pathogen borrelia burgdorferi , and is transmitted by the tick - vector ixodes scapularis . 
 it is the most prevalent arthropod - borne disease in the united states . to determine the seroprevalence of b.  burgdorferi antibodies in white - tailed deer ( odocoileus virginianus ) from texas 
 , we analyzed serum samples ( n  =  1493 ) collected during the 20012015 hunting seasons , using indirect elisa . 
 samples with higher sero - reactivity ( 0.803 and above ) than the negative control group ( 0.662 ) were further tested using a more specific standardized western immunoblot assay to rule out false positives . 
 using elisa , 4.7% of the samples were sero - reactive against b.  burgdorferi , and these originated in two eco - regions in texas ( edwards plateau and south texas plains ) . 
 however , only 0.5% of the total samples were sero - reactive by standardized western immunoblot assay . 
 additionally , both elisa and standardized western immunoblot assay results correlated with an increased incidence in human lyme disease cases reported in texas . 
 this is the first longitudinal study to demonstrate fluctuation in sero - reactivity of white - tailed deer to b.  burgdorferi sensu stricto antigens in southern united states . 
 future ecological and geographical studies are needed to assess the environmental factors governing the prevalence of lyme disease in non - endemic areas of the southern united states .