ConnieMc
Frequent Contributor (1K+ posts)
Member # 191
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I received a letter from the governor's office proclaiming May 21 to 27 tick and mosquito borne awareness week. Also $205,434 to fund a comprehensive program "to better understand the impact of tick-borne infections on the population" etc, etc. Even the funding of a nurse consultant position to assist with efforts.
Wow, I cannot believe this has come true.. Good work NCLDF. All that letter writing from the membership has paid off.
We do not have much time to prepare for the awareness week, but I will surely get some info together for my community.
Keep up the good work NCLDF!!
Posts: 2276 | From NC | Registered: Oct 2000
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char
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Yeah!
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5dana8
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Way to go Connie!!!!!!
Good luck and we all appreciate your efforts
-------------------- 5dana8 Posts: 4432 | From some where over the rainbow | Registered: Sep 2005
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I have friends who are in the allied health profession in NC. Several people are in the friends' dept. I am making information manuals for all in the dept. to help educate them and sending them a new book that was recently published on lyme. Two are married to internists. I feel obligated to help out in North Carolina- I went to graduate school there. Hope my small effort helps a little.
Posts: 719 | From Delaware | Registered: Jan 2006
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ConnieMc
Frequent Contributor (1K+ posts)
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quote:Originally posted by Aniek: Great news. Maybe publicity around this could help Dr. J?
Hopefully it will, as the first step is for the government to ADMIT that it is here. Think that has now been accomplished. Next step will be for them to realize it is not easily treated and disables people if proper treatment is not prescribed.
Dr. J's hearing is on June 14th at 8AM in Raleigh NC. As the date nears, more info will be made available for anyone who wants to attend. Meanwhile, we will take advantage of the governor's proclamation to make May 21 to 27 an awareness week. Hopefully the media will do some stories.
Posts: 2276 | From NC | Registered: Oct 2000
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Dr. J is my doc in NC, amazing person. What is happening to him is nothing more than a witch hunt.
BTW, we need to keep hitting up the N and O, they are publishing TBI research that sounds like its from the 1920's...... "the only serious threat is Rocky Mountain Spotted Fever"
Give me a break N and O, and do some fact checking.
Posts: 559 | From Cary, NC | Registered: May 2006
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ConnieMc
Frequent Contributor (1K+ posts)
Member # 191
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Lymescience, when did the N&O publish their article? I couldn't locate it online and wanted to repond. If you know it, please pass on the name of the reporter who did the story.
Reference the proclammation made by the governor. The press release is on the governor's website. I have not been successful getting interest in my area (Greensboro, W-S, High Point) from the media. What a shame. They like "scandal" and hopefully they will cover the hearing, but perhaps are not interested in educating the public about tick-borne diseses.
Posts: 2276 | From NC | Registered: Oct 2000
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Yes, I'll find that story for you, It was published in the last few weeks to warn North Carolinians about the dangers of tick bourne infections. The entire article focused on RMSF. There are a few sentences thrown in here and there about other diseases, such as HGE and STARI, but nothing that would alert the average citizen as to the danget of either of those, PARTICULARILY STARI.
Furthermore, it downplayed Lyme Disease in this region, which to my knoweledge has already been a debunked theory based upon epidemiological serveys conducted by researchers in florida who identified Borrelia Burdorferri Sensu Lato as a common occurance in the south. Furthermore, to identify this organism required a change in the testing system used. She used an amplified PCR to detect genuis Borrelia, a far more sensitive test, though not as specific. However, by running the gene codes through a national database, she was able to account for the lack of specificity.
More importantly, she HAS REPEATED THIS STUDY. It has also never been debunked. The CDC tried to debunk this study, but if you read very very carefully, they never used the same PCR primer. They used the original primer for which we already knew would show a lack of Borrelia.
Here are the links, followed by the articles incase you feel like just reading in this thread.
Journal List > Appl Environ Microbiol > v.71(5); May 2005
Abstract Full Text Figures and Tables PDF (166K) Contents Archive Journal Homepage
Related material: PubMed recordPubMed related artsPubMed LinkOutNucleotidePopsetProteinTaxonomyTaxonomy tree
PubMed articles by: Clark, K. Hendricks, A. Burge, D. Appl Environ Microbiol. 2005 May; 71(5): 2616-2625. doi: 10.1128/AEM.71.5.2616-2625.2005. Copyright � 2005, American Society for Microbiology
Molecular Identification and Analysis of Borrelia burgdorferi Sensu Lato in Lizards in the Southeastern United States Kerry Clark,* Amanda Hendricks, and David Burge
Department of Public Health, University of North Florida, 4567 St. Johns Bluff Road, Jacksonville, Florida 32224
*Corresponding author. Mailing address: Department of Public Health, University of North Florida, 4567 St. Johns Bluff Road, Jacksonville, FL 32224. Phone: (904) 620-2840. Fax: (904) 620-2848. E-mail: [email protected].
Received July 6, 2004; Accepted December 1, 2004.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Abstract Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Lyme borreliosis, the most frequently reported arthropod-borne infection in the United States (6), is caused by several species within the Borrelia burgdorferi sensu lato genogroup (38). B. burgdorferi sensu lato includes at least 11 genospecies worldwide, three of which are present in North America (Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto) (16, 28, 33). Thus far, only B. burgdorferi sensu stricto has been proven to cause human disease in the United States.
In the northeastern United States, the spirochetes are transmitted to humans by the blacklegged tick, Ixodes scapularis (5), and maintained in nature primarily by small rodents (4, 23, 31). In the southeastern and western United States, immature stages of the vector ticks feed primarily on lizards (2, 10, 35, 43). Although B. burgdorferi sensu lato has been isolated from birds, rodents, and ticks in southern and western states (9, 31, 33), the organism has never been isolated from wild lizards. Indeed, several studies have shown that strains of two B. burgdorferi sensu lato species do not survive in the blood of two lizard species found in California (19, 21, 43), leading to a widely held belief that lizards do not serve as reservoirs of the bacteria. However, a different study (22) showed in laboratory experiments that two common lizards in the southeastern United States, green anoles and southeastern five-lined skinks, were reservoir competent for one strain of B. burgdorferi sensu stricto. In the present study, we sought to determine whether lizards in the southeastern United States are naturally infected with B. burgdorferi sensu lato by attempting to isolate spirochetes and by using DNA amplification methods to genetically characterize strains present in lizards and to conduct initial experiments to determine if I. scapularis ticks could acquire B. burgdorferi sensu lato from feeding on naturally infected lizards collected in the wild. Here we present the first reported evidence of B. burgdorferi sensu lato among naturally infected wild lizards; these findings demonstrate a broad geographic distribution, three B. burgdorferi sensu lato species, and high infection prevalence among multiple lizard species in two southeastern states.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES MATERIALS AND METHODS Sample collection. Lizards were captured and sampled from national forests and state parks in northern and central Florida and southeastern South Carolina from March 2003 through May 2004. The primary habitats at the collection localities are mixed pine and oak uplands, mixed pine flatwoods, and bay swamps. Prescribed burning of vegetation is conducted regularly at some sites as part of ongoing habitat management programs. Lizards were obtained by stalking and capturing either by hand or via noosing. Attached ticks were immediately removed with forceps and preserved in ethanol. A sample (∼50 to 100 μl) of blood was obtained via tail fracture and blotted onto filter paper strips for DNA extraction. The blood from most lizards was obtained by removing the distal portion of the tail by hand, as the tails of most common lizards collected in the study fracture readily, providing a few drops of blood without harming the animals (tail fracturing is a natural escape mechanism for the animals). Most animals were returned to their site of capture immediately after examination and blood collection. Twelve broad-headed skinks (Eumeces laticeps) were euthanized humanely to harvest organs and tissues for Borrelia isolation attempts. An additional six PCR-positive E. laticeps skinks were kept in the laboratory for several months for transmission experiments.
DNA extraction and PCR testing. DNA was extracted from dried filter paper blood samples, tick pools, and cultures using a commercially available kit (MasterPure; Epicentre, Madison, WI) with optimized modifications of the manufacturer's protocols for each starting material. The starting template for filter paper blood samples was an approximately 5- by 5-mm square piece of blood-soaked paper. Culture aliquots of 200 μl were taken from approximately the middle of each conical tube of 4 ml of media suspension in attempts to avoid obtaining dead spirochetes that would presumably settle to the bottom of the tubes. The resulting DNA pellets for all extracts were rehydrated in 100 μl of Tris-EDTA buffer. Negative control samples free of any template were included in each round of DNA extractions. Extracts were first screened by nested PCR targeting a 389-bp portion of the conserved 41-kDa chromosomal flagellin gene (flaB) of B. burgdorferi sensu lato using slight modifications of published primers (Table 1) (15, 32, 42). All flaB-positive samples were further tested with a nested PCR assay targeting a 236-bp portion of the chromosomal 66-kDa protein (p66) using a combination of published primers (36) and one that we modified (Table 1). The same subset of flaB-positive samples was tested with primers (13) targeting a 352-bp portion of the genetically diverse outer surface protein A (ospA) gene of B. burgdorferi sensu lato.
First-round amplifications contained between 2.5 and 5 μl of DNA extract per individual sample in a total reaction volume of 50 μl. All reactions utilized a hot start master mix (HotMasterMix; Brinkmann-Eppendorf, Westbury, NY), resulting in a final concentration of 1.0 U of Taq DNA polymerase, 45 mM KCl, 2.5 mM MgCl2, 200 μM concentrations of each deoxynucleoside triphosphate, and a 0.5 μM concentration of each primer, and were carried out in an automated DNA thermal cycler (PTC 200; MJ Research, Watertown, MA). Outer reaction PCRs consisted of initial denaturation at 95�C for 1 min followed by 40 cycles of 94�C for 30 s, primer annealing at the temperature listed in Table 1 for 30 s, and extension at 68�C for 45 s. Nested reactions included between 1 and 2.5 μl of outer reaction product as template for another 30 cycles with the same parameters and annealing temperature profile as described above and in Table 1.
PCRs were set up in a separate area within a PCR clean cabinet (CleanSpot Workstation; Coy Laboratory Products, Grass Lake, MI) equipped with a germicidal UV lamp. Other precautions to prevent carryover contamination of amplified DNA included different sets of pipettes dedicated for DNA extraction, PCR setup, and postamplification activities; the use of aerosol barrier filter pipette tips; and exposing PCR tubes, pipettes, and tips to UV light prior to PCR setup. Each outer PCR included a negative control sample with sterile water as template and a positive control sample from B. burgdorferi sensu stricto strain B31 culture extract. Negative control samples included in each round of extractions were also screened to detect any possible extraction contamination. A portion of the positive and negative control outer reaction samples was carried over as template in each nested reaction, just as for experimental samples. PCR products were electrophoresed in 2% agarose gels, which were stained with ethidium bromide, and visualized and recorded with a digital gel documentation unit.
DNA sequence analysis. PCR products were purified using the MinElute PCR purification kit (QIAGEN, Valencia, CA). DNA templates were sequenced using the fluorescent dideoxy terminator method of cycle sequencing on either a Perkin-Elmer, Applied Biosystems, Inc. 373A or 377 automated DNA sequencer following Applied Biosystems protocols (29). Sequences were generated using the Sequencher Software (Gene Codes Corporation, Ann Arbor, MI). Investigator-derived sequences were compared with those obtained by searching the GenBank database (National Center for Biotechnology Information) using the Basic Local Alignment Search Tool (BLAST) (1) and aligned using Clustal X (40). Phylogenetic trees were constructed using the neighbor-joining (NJ) and maximum parsimony (MP) methods (37, 39) with the tree-building program MEGA version 2.1 (18). Tree topologies and genetic relationships obtained with the two methods were compared for consensus. To estimate node reliability of trees obtained with each method, bootstrap values (11) based on an analysis of either 100 (MP) or 1,000 (NJ) replicates were determined. Pairwise distances were computed by the Kimura two-parameter model (17).
Borrelia culture isolation. We chose 12 flaB PCR-positive broad-headed skinks (nine males and three females) for attempts to isolate spirochetes. Within 1 week of testing PCR positive, the animals were euthanized humanely and dissected with sterile technique inside a biosafety cabinet. Whole blood (∼100 μl), heart, and liver samples (and testes from males; ∼50 mg of each tissue type per sample) were inoculated into tubes containing 4 ml of modified Barbour-Stoenner-Kelly (BSK-H) culture medium supplemented with antibiotics (Sigma, St. Louis, MO) for isolating Borrelia (3), incubated at 32�C, and examined weekly for spirochetes by dark-field microscopy for 8 weeks. Samples from B. burgdorferi sensu lato reference strains (B. andersonii MOK-1c; B. bissettii 25015; B. burgdorferi sensu stricto B31, JD1, NC92, and WI90; and Borrelia sp. strain SCW-30h) were also inoculated and maintained in culture to ensure the ability of the medium to support spirochete growth.
Tick-feeding experiments. Six additional broad-headed skinks (three each from Florida and South Carolina) that tested positive for B. burgdorferi sensu lato in initial PCR tests were chosen for a tick-feeding and infection experiment. Individual animals were placed inside a ∼1.5-in.-diameter tube constructed of 1/4-in.-mesh hardware cloth, with PVC end caps modified from published designs (14, 22). Approximately 100 I. scapularis tick larvae from a laboratory-reared colony at Colorado State University were introduced to each lizard's tube. Feeding tubes were suspended over white plastic photo trays with wet paper towel substrate in separate aquaria. The rims of the trays and aquaria were coated with petroleum jelly to prevent escape of ticks that dropped off. Trays were checked daily for detached ticks. Lizards were fed one or two crickets every other day by removing the PVC end cap on one end of the tube. Water was provided via a moistened cotton ball kept inside the tube at all times. After blood-fed ticks dropped off, they were rinsed in a 5% bleach solution, followed by a rinse in sterile distilled water, and then maintained in glass vials with cloth mesh lids in a glass desiccator jar at ∼97% rH and 82�F until they molted to nymphs (∼1 month later). The resultant nymphs were tested by PCR to determine if they acquired B. burgdorferi sensu lato and maintained infection through the molt. All procedures involving capturing, maintaining, and testing lizards were conducted in accordance with protocols approved by the University of North Florida Institutional Animal Care and Use Committee.
Nucleotide sequence accession numbers. The GenBank accession numbers for the B. burgdorferi sensu lato flaB gene sequences generated in the present study are AY662999 to AY663008, AY663010 to AY663017, AY823241, AY823242, and AY823244 to AY823249. The B. burgdorferi sensu lato ospA gene sequences reported here are AY663018 to AY663021, AY663023, AY663024, AY823229, AY823231, and AY823232. The B. burgdorferi sensu lato p66 gene sequences reported here are AY823233 to AY823240. The sequences used for comparison are listed in Table 2.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES RESULTS B. burgdorferi sensu lato-specific PCR. We captured and obtained blood samples from 160 lizards representing seven genera and 10 species from sites in Florida and South Carolina. Most (77%) of the animals were captured in March and April of 2003 and 2004, which is before most I. scapularis larvae and nymphs become active, or during the period of initial activity in the spring (35). Most (93%) animals were free of ticks when they were captured. Only 18 I. scapularis larvae and 30 nymphs were observed and removed (all during April and May), and all came from three lizard species: broad-headed and southeastern five-lined skinks and eastern glass lizards.
To determine if the lizards' blood contained B. burgdorferi sensu lato DNA, we first screened blood sample extracts with the B. burgdorferi sensu lato-specific nested flaB PCR assay. Eighty-six (53.8%) of the samples tested positive, including those from nine lizard species (six genera) and nine sites in Florida and South Carolina (Table 3); positive blood samples were obtained from juvenile, subadult, and adult animals. None of the negative control extracts tested positive. The infection prevalence among different positive lizard species ranged from 18 to 100%. The prevalence among animals from South Carolina (72%) was higher than for those from Florida (40%), but a greater variety of species from Florida was tested. The only species for which no positives were obtained was the Mediterranean gecko, which, along with two brown anoles that did test positive, came from suburban residences. Positive results were obtained for several species that are not commonly, if ever, parasitized by I. scapularis: brown and green anoles, Florida scrub lizards, and six-lined racerunners (2, 10). flaB PCR-positive samples were obtained from lizards in February through May, September, and December, which includes months (February and December) when I. scapularis larvae and nymphs do not typically parasitize hosts (35).
All 86 flaB-positive samples were then tested with the p66 and ospA nested PCR assays. Thirty (34.9%) of these 86 tested positive with the p66 assay, and 17 (19.8%) were also positive with the ospA PCR assay. Only 10 of 86 (11.6%) flaB-positive samples tested positive with both p66 and ospA assays. Most PCR-amplified target gene fragments were visible in ethidium bromide-stained gels only in the nested reaction products, indicating generally low target copy number in the extracts. Nevertheless, many lizard blood extracts were tested several times for each of the three target gene fragments, with consistent results. Some samples were positive only with the flaB assay. Some were consistently flaB and p66 positive but ospA negative, and others were consistently flaB and ospA positive but p66 negative.
B. burgdorferi sensu lato flaB, p66, and ospA sequences. We sequenced PCR-amplified flaB, ospA, and p66 gene fragments from many individual lizard samples and compared the sequences to those obtained via BLAST (1) searching the GenBank database. Most of the flaB and ospA DNAs analyzed in this study were sequenced in one direction only, with the forward primers used in the PCRs. A few of the flaB DNAs and all of the p66 DNAs were sequenced in both directions with the forward and reverse PCR primers. For a few samples, the initially derived sequences contained some unresolved nucleotides caused by multiple signal polymorphisms at those positions. When these were resequenced, or the sequences obtained with both forward and reverse primers were compared, the unresolved bases in all but a few of these samples could be determined. Based on the BLAST scores and phylogenetic comparisons we conducted, all flaB, ospA, and p66 DNAs obtained from lizards or I. scapularis ticks fed upon them in the lab belonged to B. burgdorferi sensu lato.
Figure 1 shows an NJ phylogenetic tree based on 362 bp of the flaB gene, which has been used to reliably differentiate B. burgdorferi sensu lato strains to the species level (12, 15, 32). flaB sequences derived from 28 lizards and four I. scapularis nymph pools (from larvae fed on infected lizards in the lab) clustered into three clades defined by reference strains of the B. burgdorferi sensu lato species previously identified in the United States; these included 19 B. burgdorferi sensu stricto strains in eight lizard species and the four I. scapularis nymph pools, 11 strains of B. andersonii in five lizard species, and 2 B. bissettii strains in two lizard species (Fig. 1; Table 4; not all data shown). Based on pairwise distances, the flaB sequences from lizards and ticks that clustered with B. burgdorferi sensu stricto strains were between 98.0 and 100% identical and between 98.6 and 100% similar to the B. burgdorferi sensu stricto reference strains included in the analysis. flaB sequences from several other lizards clustered with those from B. andersonii reference strains (Fig. 1). All except one (SC194) of these sequences from the lizards were nearly identical to that for strain SI-10, a B. andersonii isolate from a blacklegged tick in Georgia. The similarity among all of the B. andersonii flaB sequences ranged between 98.0 and 100%. The lizard flaB sequences from FL60 and FL203 were 99.7% identical and between 98.9 and 99.7% identical to B. bissettii reference strain sequences included in the comparison.
The ospA sequences (334 bp analyzed) derived from lizards clustered into three different clades defined by the previously described North American strains (Fig. 2). The sequences were heterogeneous, and their phylogenetic clustering did not agree with that obtained from the flaB sequence analysis for several individual lizard templates. For example, the flaB sequence from lizard samples SC89, SC167, and FL187 clustered with B. andersonii strains (Fig. 1), but the ospA sequences from these samples clustered with B. bissettii sequences (Fig. 2). The FL121 and FL126 flaB sequences both clustered with B. burgdorferi sensu stricto strains (Fig. 1; FL126 not shown), yet while the FL121 ospA sequence clustered along with strain SCW-30h most closely to B. burgdorferi sensu stricto reference strain sequences, the FL126 ospA sequence clustered with B. bissettii strains.
The ospA sequences from lizards FL126, FL187, SC89, SC167, and SC194 were between 99.1 and 100% identical and between 93.4 and 100% similar to all B. bissettii reference strain sequences included in the comparison and most similar to previously described B. bissettii strains from the southeastern United States. The ospA sequence from lizard FL121 was 99.7% similar to that for strain SCW-30h and between 93.6 and 95% similar to B. burgdorferi sensu stricto reference strains included in the analysis. The sequences from lizards SC87, SC106, and SC107 were 99.7 to 100% identical and between 97.2 and 98.7% similar to the B. andersonii reference strains.
All of the p66 sequences derived from lizards were also very similar to B. burgdorferi sensu lato reference strains. The phylogenetic tree based on 233 bp of data showed that all but one of the lizard-derived p66 sequences analyzed in this study clustered most closely with B. burgdorferi sensu stricto reference strains (Fig. 3). The phylogenetic placement of some lizard p66 sequences also did not correlate with the B. burgdorferi sensu lato species clustering produced by the flaB sequence analysis for those lizard samples. The sequences from lizards FL71, FL118, FL131, FL139, FL187, SC87, and SC194 were 98.3 to 100% identical to each other as well as to the B. burgdorferi sensu stricto reference strains that they were most similar to. The p66 sequence from lizard SC152 was 99.6% identical to that from B. bissettii strain 25015, 98.3% identical to that from FL187, 97.6% identical to that from B. bissettii IS-19 from Colorado, and 96.8% identical to that from B. andersonii MOK-1c.
Borrelia isolation. Although all lizard blood and tissue cultures were examined weekly for spirochetes for 8 weeks, no spirochetes were detected microscopically. During that time, we extracted and tested DNA from cultures of different starting materials at different times postinoculation using the nested flaB PCR assay. At least one template type culture sample from each of the 12 animals tested positive via PCR. Target products were seen only in the nested reactions, indicating a very low copy number of target DNA template in the culture extracts. One of 12 whole-blood cultures and 1 of 12 liver tissue cultures tested at 1 week postinoculation were PCR positive. At 4 weeks postinoculation, 7 of 12 blood culture extracts tested positive. At 5 weeks, 5 of 12 heart tissue cultures and 3 of 9 testes cultures were positive. However, upon testing again at 8 weeks, only 2 of 12 new extracts from previously positive culture samples (seven blood samples, three heart samples, and two testes samples) were positive. Most of the lizard samples were visibly contaminated with other bacteria. PCR amplification of lizard blood and tissue culture DNA extracts using broad-range 16S rRNA gene primers and DNA sequencing confirmed the presence of Mycoplasma spp. Reference strain cultures of several B. burgdorferi sensu lato species grew extremely well and did not become contaminated. During the follow-up, flaB PCR-positive lizard culture samples were subcultured multiple times into fresh medium; however, the contaminating bacteria persisted and continued to grow in the medium.
Tick-feeding experiments. In August 2003, blood sample extracts from 9 of 20 (45%) broad-headed and southeastern five-lined skinks that had been kept in the laboratory since being captured in April of that year tested positive via flaB PCR, and 6 of these (three broad-headed skinks each from Florida and South Carolina) were used in the tick-feeding and transmission experiment. Approximately 100 I. scapularis larvae were placed on each animal, and 393 attached and fed to repletion. The minimum and maximum numbers that fed on different lizards were 11 and 124, respectively, with an average of 66 per animal. Blood-fed ticks were kept in the lab until they molted to nymphs. Unfortunately, due to fungal growth in the tubes, only 28 ticks survived through the molt, which was deemed too few to attempt further transmission studies. However, DNA was extracted from the nymphs in six pools of three to six ticks per pool to aim for maximum sensitivity of detection and tested by nested flaB PCR to determine whether they acquired B. burgdorferi sensu lato and maintained detectable B. burgdorferi sensu lato DNA through the molt. All six pools tested positive, indicating a minimum estimated ``infection'' prevalence of 6 of 28 (21%); the actual number of infected individual ticks may have been higher. The flaB products were sequenced from four of the pools. They were not identical, but all were found to be >99% similar to lizard-derived and reference strain B. burgdorferi sensu stricto sequences as described above.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES DISCUSSION Studies have shown that two lizard species in California, the western fence lizard (Sceloporus occidentalis) and the southern alligator lizard (Elgaria multicarinata), have poor reservoir potential for some strains of B. burgdorferi sensu lato (19, 20, 21, 41, 43). Another study (22) showed that green anoles and southeastern five-lined skinks, both common in the southeastern United States, were reservoir competent under laboratory-controlled conditions for one strain of B. burgdorferi sensu stricto. In the present study, we were unable to obtain pure cultures of B. burgdorferi sensu lato from wild caught lizards, including those containing strains belonging to B. burgdorferi sensu lato species that have been cultured in BSK medium. We did, however, demonstrate the presence of B. burgdorferi sensu lato DNA in cultures of different sample types by PCR testing up to 8 weeks postinoculation.
These results may be explained by the fact that culturing in BSK medium is selective for specific genotypes of B. burgdorferi sensu lato (26, 30). Oliver et al. (31) found relatively low Borrelia culture isolation prevalence (6.5%) among 200 specimens of three reservoir-competent small mammal species sampled from several sites in Florida. Attempts to isolate spirochetes in BSK medium from a smaller number (n = 65) of small mammals from northeast Florida in a different study conducted by one of us (K. Clark) also failed, even though PCR testing showed the B. burgdorferi sensu lato prevalence to be very high (7). An alternative explanation for the failure to isolate B. burgdorferi sensu lato from lizards in the present study is that the number of spirochetes present in the lizard blood and tissues may have been below the sensitivity level of this detection method, especially if the spirochetes were competing with other bacteria that may have been present in the samples. Additional attempts at isolating spirochetes from more PCR-positive lizards are necessary to determine whether such strains are cultivable in BSK medium. These efforts could include filtering inoculated cultures in attempts to remove bacteria other than spirochetes that may be present in lizard blood and tissues. Attempting to isolate these Borrelia organisms in tick cell lines is another potentially successful strategy.
Enzootic cycles of B. burgdorferi sensu lato transmission in the southeastern United States are more complex than those in the northeastern United States due to the involvement of a greater number of vertebrate and tick species in the South (2, 8, 31). This ecologic diversity may explain the genetic heterogeneity among southern B. burgdorferi sensu lato strains and the variation between southern strains and those found elsewhere (24, 25). This heterogeneity may explain why the p66 and ospA primers we used did not amplify products from some of the flaB-positive samples we tested. Primer mismatch could have led to a complete lack of amplification or simply reduced the sensitivity of the PCR assays below the level needed for detection. Numerous strains of B. burgdorferi sensu lato were described previously from ticks and small mammals in Florida by using similar techniques (7). Many of the B. burgdorferi sensu stricto flaB-positive samples in that study also failed to test positive with primers designed to amplify portions of several genes, including p66 and ospA, indicating either low target gene copy number in the samples or primer mismatch. BSK culture attempts with human tissue and fluid samples also fail to isolate spirochetes from many samples that test positive via PCR assays. Interestingly, the p66- and ospA-positive prevalences among flaB-positive lizards in the present study (34.9 and 19.8%, respectively) are comparable to the p66 and ospA PCR-positive prevalences among human spinal fluid (39 and 23%, respectively) and urine (42 and 24%, respectively) samples from Lyme arthritis patients in one study (34).
Based on the findings of several studies (12, 15, 32), we considered the phylogenetic clustering of lizard-derived B. burgdorferi sensu lato strains from partial sequences of the highly conserved flaB gene as indicative of species-specific groupings. Our analyses showed much more variability in the partial ospA and p66 gene sequences derived from lizards. For some templates, the ospA and p66 sequences were very similar to those of reference strains of the same species. For other animals, however, the ospA and p66 sequences derived from them clustered with reference strains of a different species group. There are several possible explanations for this. There may be significant genetic variability in the ospA and p66 genes of southern B. burgdorferi sensu lato strains. This might include, for example, genetic exchange of the plasmid ospA gene among strains of different species, similar to that shown for ospC (27). It is also possible that multiple B. burgdorferi sensu lato species were present in the blood of many individual lizards, representing a relatively high rate of multiple infection. Additional testing of our samples with an adequately sensitive, PCR-based method that employs B. burgdorferi sensu lato species-specific DNA probes could determine this.
Our DNA sequencing did produce some unresolved bases indicative of possible polymorphisms at some locations in the flaB and p66 sequences from a small number of animals. Most of these were resolved upon resequencing those templates with the same primers or the complementary primers used in the PCRs. However, in the flaB sequences for a couple of samples, polymorphisms were still evident, and these were located at positions where nucleotide variation occurs between B. andersonii and B. burgdorferi sensu stricto strains.
We took extensive measures to prevent and identify any contamination of our DNA samples. There was no evidence of contamination of our DNA extracts with reference strain DNA. We used a B. burgdorferi sensu stricto reference strain (B31) as a positive control in our PCR assays, and although we did identify several B. burgdorferi sensu stricto strains in lizards, none of their flaB, ospA, or p66 sequences were identical to those for B31. The significant genetic variability among the lizard-derived B. burgdorferi sensu lato strains, compared to B. burgdorferi sensu lato reference strains of B. andersonii, B. bissettii, and B. burgdorferi sensu stricto, therefore renders contamination from reference strains a very unlikely explanation of our findings.
In the present study, we confirmed the presence of DNA of three B. burgdorferi sensu lato species in over 50% of tested lizards representing several genera and nine species from Florida and South Carolina. We detected the spirochetes in some lizards sampled during months of the year when I. scapularis larvae and nymphs do not normally parasitize hosts, and the duration of the PCR-positive status of several lizards kept in the lab exceeded 4 months. Furthermore, I. scapularis nymphs from larvae that fed on PCR-positive lizards also tested positive for B. burgdorferi sensu lato via PCR, evidence that the ticks acquired the bacteria from the lizards and maintained them after molting to nymphs. All of these findings suggest, but do not prove, the presence of live spirochetes in these samples.
If lizards serve as reservoirs of B. burgdorferi sensu lato, two factors affecting their significance as such would be the duration of infection and their longevity. The duration of B. burgdorferi sensu lato infection of lizards in the wild is not known, and reliable estimates of lizard longevity in the wild are difficult to find in the published literature. However, one website that contains information provided by 234 institutions and 425 private collections (F. Slavens and K. Slavens, Reptiles and amphibians in captivity--longevity--home page. Last updated 20 March 2003. Retrieved 8 November 2004. http://www.pubmedcentral.gov/redirect3.cgi?&&reftype=extlink&artid=1087528&iid=117184&jid=83&&http://www.pondturtle.com/longev.html) provides longevities of captive specimens of many species. The figures provided for representatives of the species included in the present study ranged from approximately 5 to 7 years for green anoles, 2 to 7 years for various Eumeces spp. skinks, 1 to 4 years for Cnemidophorus spp., 8 years for the Mediterranean gecko (Hemidactylus turcicus), 3 to 14 years for the eastern glass lizard (Ophisaurus ventralis), 1 to 8 years for the eastern fence lizard (Sceloporus undulatus) and other Sceloporus spp., and 2 years for the ground skink (Scincella lateralis). These figures agree with those provided at another site that contains detailed information about brown and green anoles (Under the leaves: complete anole care. Last updated 23 July 2003. Retrieved 8 November 2004. http://www.pubmedcentral.gov/redirect3.cgi?&&reftype=extlink&artid=1087528&iid=117184&jid=83&&http://www.kingsnake.com/anolecare/5.htm). Although it is doubtful that most specimens live as long in the wild, it is possible that many wild specimens live for several years.
The minimal number of ticks removed from the lizards in this study, the distribution of infection among animals of different ages (including juveniles), and the diversity of infected lizard species, including some not commonly parasitized by ticks and a few individuals (brown anoles) from residential areas, all suggest the possibility of alternate transmission pathways besides the tick-borne route. Some other possible means of transmission to be investigated include transmission by mosquitoes, flies, or mites through blood feeding or mechanical transmission; by lizards ingesting infected arthropods; and from lizard to lizard vertically via transovarial transmission and horizontally during mating.
The present study's findings show that lizards in the southeastern United States are naturally infected with Lyme borreliae and suggest that they may play a role in the enzootic maintenance of B. burgdorferi sensu lato in the region. It remains to be conclusively shown whether the strains infecting lizards in the southeastern United States are cultivable in BSK medium and infectious or pathogenic to humans and whether ticks or other hematophagous arthropods acquire and maintain viable B. burgdorferi sensu lato spirochetes from feeding on lizards. Therefore, the relevance of this discovery to human disease risk in the region is not yet known.
Acknowledgments We thank J. F. Piesman, Centers for Disease Control and Prevention, Fort Collins, CO, and J. H. Oliver, Georgia Southern University, Statesboro, GA, for providing reference strain culture samples. We also thank H. J. Hutcheson, Colorado State University, Fort Collins, CO, for providing colony ticks for the transmission experiments.
This work was supported in part by a grant from the American Lyme Disease Foundation, Somers, NY, and a University of North Florida College of Health Dean's professorship award to K.C. funded by the Brooks Health Foundation, Jacksonville, FL.
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Figures and Tables FIG. 1. Unrooted neighbor-joining phylogenetic tree based on 362 bp of the flaB gene obtained from lizards in Florida and South Carolina and B. burgdorferi sensu lato reference strains. The relapsing fever group species Borrelia lonestari was included as an outgroup. (more ...) FIG. 2. Unrooted maximum parsimony bootstrap consensus phylogenetic tree based on 334 bp of the ospA gene obtained from lizards in Florida and South Carolina and B. burgdorferi sensu lato reference strains. Numbers at the branch nodes represent bootstrap values (more ...) FIG. 3. Unrooted neighbor-joining bootstrap consensus phylogenetic tree based on 233 bp of the p66 gene obtained from lizards in Florida and South Carolina and B. burgdorferi sensu lato reference strains. The relapsing fever group species Borrelia hermsii was (more ...) TABLE 1. Oligonucleotide primers used in this study TABLE 2. Borrelia species reference strains used in this study TABLE 3. Prevalence of B. burgdorferi sensu lato flagellin (flaB) gene DNA among lizards from Florida and South Carolina TABLE 4. Representative Lyme borreliosis group spirochete strains identified in the present study based upon phylogenetic analysis of flaB, ospA, and p66 gene sequencesa
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PubMed articles by: Clark, K. J Clin Microbiol. 2004 November; 42(11): 5076-5086. doi: 10.1128/JCM.42.11.5076-5086.2004. Copyright � 2004, American Society for Microbiology
Borrelia Species in Host-Seeking Ticks and Small Mammals in Northern Florida Kerry Clark*
Department of Public Health, University of North Florida, Jacksonville, Florida
*Mailing address: Department of Public Health, University of North Florida, 4567 St. Johns Bluff Rd., Jacksonville, FL 32224. Phone: (904) 620-2840. Fax: (904) 620-2848. E-mail: [email protected].
Received February 11, 2004; Revised June 24, 2004; Accepted July 4, 2004.
This article has been cited by other articles in PMC. Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Abstract The aim of this study was to improve understanding of several factors related to the ecology and environmental risk of Borrelia infection in northern Florida. Small mammals and host-seeking adult ticks were collected at several sites, and specimens were tested for the presence of Borrelia species, primarily by PCR amplification. Tissues from some vertebrates and ticks were initially cultured in BSK-H medium to isolate spirochetes, but none were recovered. However, comparison of partial flagellin (flaB), 66-kDa protein (p66), and outer surface protein A (ospA) gene sequences from DNAs amplified from small mammals and ticks confirmed the presence of several Borrelia species. Borrelia lonestari DNA was detected among lone star ticks (Amblyomma americanum) at four sites. Borrelia burgdorferi sensu stricto strains were detected in all small mammal species tested and in A. americanum, Ixodes affinis, and Ixodes scapularis ticks. Borrelia bissettii was found in a cotton mouse and cotton rats and in I. affinis ticks. The study findings extend the known geographic distributions of B. lonestari in A. americanum and of B. burgdorferi sensu lato in A. americanum, I. affinis, I. scapularis, and small mammals to new sites in Florida. The presence of B. burgdorferi sensu stricto strains in host-seeking lone star ticks at two sites in Florida suggests that A. americanum should still be considered a possible vector of B. burgdorferi sensu lato.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Lyme disease (LD), the most prevalent arthropod-borne disease in the United States, is caused by several species of spirochete bacteria within the Borrelia burgdorferi sensu lato genogroup (19). These species are maintained in nature and transmitted to humans by ticks of the genus Ixodes. Clinical symptoms affect all age groups and may involve the skin, joints, nervous system, and heart (49). B. burgdorferi sensu lato includes at least 10 genospecies, 3 of which (B. burgdorferi sensu stricto, Borrelia andersonii, and Borrelia bissettii) are present in North America (27, 41). In the northeastern United States, B. burgdorferi sensu stricto is the most common species, but it also occurs in western and southern states (28, 36, 41). Only B. burgdorferi sensu stricto has been proven to cause human disease in the United States. However, some B. bissettii-like strains may also be pathogenic (39, 51). There is significant genetic diversity among B. burgdorferi sensu lato strains and species in North America, particularly in areas other than the Northeast (4, 26, 28).
Most of what is known about the ecology of B. burgdorferi sensu lato in the eastern United States was derived from studies conducted in the Northeast, where the majority of human cases have occurred. There, B. burgdorferi sensu stricto is transmitted to humans by the blacklegged tick, Ixodes scapularis (6), and maintained in nature primarily by the white-footed mouse (Peromyscus leucopus) (25). Nevertheless, since the disease has become reportable, hundreds of cases have been reported from southeastern states, including Florida, Georgia, and South Carolina (9). Some of these cases may have resulted from exposures that occurred elsewhere in the country; however, many were locally transmitted (33). B. burgdorferi sensu lato (including B. burgdorferi sensu stricto) has been isolated from birds, rodents, and ticks in Florida, Georgia, South Carolina, and other southern states (12, 34, 36), but despite the information gathered to date, it is still unclear whether endemic human infection with B. burgdorferi sensu lato commonly occurs in the southern United States. Although LD incidence rates show that human risk is significantly lower in the Southeast than the Northeast, the underlying reasons for this are not well understood.
Complicating our understanding of LD in the Southeast is the emergence of a southern tick-associated rash illness (STARI) resembling the presentation of LD (8, 18, 23). STARI, also known as Master's disease, is associated with bites from the lone star tick, Amblyomma americanum (18, 23). Lone star ticks from several states in the eastern United States contained spirochetes that were noncultivable in BSK medium, which is typically used to isolate B. burgdorferi sensu lato, and are more closely related to relapsing fever Borrelia species (5, 7, 50, 52). This spirochete was named Borrelia lonestari (5).
The purpose of this study was to improve understanding of several factors related to the ecology and environmental risk of Borrelia infection in a large region of northern Florida. Specific objectives were to clarify the presence and distribution of Borrelia species among host-seeking ticks and small mammals; to identify, investigate genetic variability of, and characterize Borrelia strains by using molecular techniques; and to estimate the prevalence of infection with distinct Borrelia species among ticks and small mammals at selected study sites.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES MATERIALS AND METHODS Study area and localities. Ticks were collected primarily at public recreation areas (state parks, wildlife management areas, and national forests) located in the northeastern region of Florida. The major terrestrial habitat types at those sites are pine flatwoods, mixed hardwood forest, coastal maritime hammock, high pine, and scrub (31). Small mammal sampling was conducted at two sites. The University of North Florida Wildlife Sanctuary (UNFWS), located on the main campus in southeast Jacksonville, is a fragmented island of several hundred acres of natural habitat surrounded by development. The primary habitats are mesic to hydric mixed pine flatwoods (dominated by longleaf and slash pine, loblolly bay, and saw palmetto) and xeric to mesic mixed pine and oak uplands (dominated by longleaf and slash pine, turkey and live oak, scrub oak, and saw palmetto). The Guana River State Park and Wildlife Management Area (GRSPWMA) is located 30 miles south of Jacksonville and is a larger area (several thousand acres) bordered by the Atlantic Ocean on the east and by residential development on the west. It contains diverse and abundant wildlife and three distinct habitats: xeric to mesic maritime hammock (live oak, hickory, pines, holly, magnolia, and saw palmetto), mesic to hydric mixed pine flatwoods, and xeric to mesic mixed oak scrub (live oak, other oaks, and saw palmetto).
Vertebrate and tick sampling. Small mammals were captured live in Sherman traps baited with wild birdseed set in line transects in different habitat types at UNFWS and GRSPWMA between April and September 1999 and from July through September 2000. Captured animals were anesthetized by ketamine hydrochloride-xylazine injection, weighed, measured, and sexed. Ectoparasites were removed and preserved in ethanol for identification as part of a related study. A sample (∼100 μl) of whole blood was collected via tail clip on Nobuto filter paper strips (Advantec MFS, Inc., Pleasanton, Calif.), allowed to dry, and stored under refrigeration until used for DNA extraction. The ears of captured animals were moistened with 70% ethanol and allowed to dry prior to removal of three 2-mm punches of tissue from each ear by using a rodent ear tag punch. Punches from one ear were placed in a 70:30 solution of sterile phosphate-buffered saline-glycerol and stored frozen for DNA extraction. Other samples were stored under refrigeration for no more than a few days prior to use in Borrelia isolation attempts. After examination and full recovery, animals were returned to their capture site. All procedures involving trapping and sampling of vertebrates were conducted in accordance with guidelines approved by the University of North Florida Institutional Animal Care and Use Committee and with permits from the Florida Department of Environmental Protection and Fish and Wildlife Conservation Commission.
Host-seeking ticks were collected by dragging 1-m2 white felt flags along vertebrate trap transects, nature trails, and firebreaks at numerous study sites and removing ticks from clothing and the drag every ∼15 m (every 15 to 20 paces). Most ticks were stored in ethanol for DNA extraction. Ticks destined for culture isolation were maintained live in vials with a few blades of fresh grass.
Borrelia isolation. Attempts were made to isolate Borrelia from some vertebrate ear tissue samples and adult blacklegged ticks. Three ear punches from each rodent were removed from their transport vial; rinsed briefly in 10% povidone iodine, then in 70% ethanol, and then twice in sterile water; air dried; and finally placed in 4 ml of fresh BSK-H complete medium (Sigma, St. Louis, Mo.) supplemented with antibiotics (3). Live ticks were likewise surface sterilized, placed in a microtube with 200 μl of fresh medium, and ground with a sterile disposable pestle. Half of the suspension was inoculated into a fresh tube with 4 ml of medium. The other half was frozen for DNA extraction to compare the sensitivity of culture with that of DNA amplification. Samples from B. burgdorferi sensu lato reference strains (B. burgdorferi sensu stricto B31, JD1, NC92, and WI90; Borrelia sp. strain SCW-30H; and B. andersonii MOK-1C) were also inoculated to ensure the ability of the medium to support spirochete growth. Cultures were incubated at 33�C and examined for spirochetes by dark-field microscopy weekly for 4 weeks.
DNA extraction. All DNA extractions were conducted within a class II biological safety cabinet (NuAire, Plymouth, Minn.) used only for this purpose. DNA was extracted from host-seeking ticks, vertebrate ear tissue punches, Nobuto blood samples, and culture samples by using the DNeasy tissue kit (Qiagen, Valencia, Calif.) with optimized modifications of the manufacturer's protocols for each starting material. Early in the study, some ticks of the same species from the same site were pooled for DNA extraction. Later, DNA was extracted from individual ticks. The amounts of template typically used for other sample types were two to three 2-mm ear punches from an individual animal, a 5- by 5-mm piece of blood-soaked Nobuto strip from an individual animal, or a 1-ml sample of culture. All samples were incubated in 100 μg of proteinase K-tissue lysis buffer; ticks and ear punch samples were incubated at 55�C overnight, and Nobuto fragment and culture samples were incubated for a minimum of 1 h. After binding to the spin column and washing twice, DNA was eluted from the columns in a final volume of 200 μl of buffer AE for all samples, including those for pooled or individual ticks.
PCR testing. Due to low Borrelia target gene copy numbers in the extracts from vertebrates and ticks and to extraneous products in single-reaction PCRs with a high number of cycles, DNA extracts from ticks and from animal tissue and blood samples were tested for most Borrelia target genes via nested PCR assays. Several primer sets for different genes were used (Table 1).
Lone star tick extracts were first tested with a nested PCR assay designed to amplify a portion of the highly conserved 41-kDa chromosomal flagellin (flaB) gene of Borrelia species (5). Flagellin-positive samples were then confirmed by testing with a Borrelia species-specific 16S rRNA gene (rDNA) primer set (42). Samples were initially screened for the presence of B. burgdorferi sensu lato DNA by using a different nested PCR designed to amplify a portion of the flaB gene of all B. burgdorferi sensu lato species (20). Most of the samples that tested positive in this assay were then tested with a nested PCR assay that amplifies a portion of the chromosomal 66-kDa protein (p66) gene of B. burgdorferi sensu lato in the United States (44). For this assay, a different inner reverse primer (p66 inner 2 [Table 1]) was designed and used to amplify a 296-bp product. Many of the B. burgdorferi sensu lato flaB-positive samples were also tested with two nested PCR assays that target portions of the ∼31-kDa outer surface protein A (ospA) gene of B. burgdorferi sensu lato (16, 17) and with a nested assay with primers targeting the intergenic spacer region (ISR) between the rrf (5S)-rrl (23S) rDNA (43).
Reaction mixtures for single-stage PCRs and first-round amplifications of nested PCR assays contained between 2.5 and 5 μl of DNA extract per individual sample in a total reaction volume of 50 μl. Extracts from individual ticks from some sites were initially screened in pools of three for efficiency. All reactions utilized a hot start master mix (TaKaRa Taq HS; PanVera Corp., Madison, Wis.), resulting in final concentrations of 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 200 μM each deoxynucleoside triphosphate, 1.25 U of Taq polymerase, and 0.5 μM each primer, and were carried out in an automated DNA thermal cycler (Geneamp PCR System 2400 [Perkin-Elmer, Norwalk, Conn.] or PTC 200 [MJ Research, Watertown, Mass.]). Single-stage and outer PCRs consisted of initial denaturation at 95�C for 1 min, followed by 40 cycles of 94�C for 30 s, primer annealing at the temperature listed in Table 1 for 30 s, and extension at 72�C for 1 min. Mixtures for nested reactions included between 1 and 2.5 μl of outer reaction product as the template for another 30 cycles with the same parameters and annealing temperature profile as described above and in Table 1.
PCRs were set up in a separate area within PCR clean cabinets (CleanSpot workstation [Coy Laboratory Products, Grass Lake, Mich.] or PCR workstation [CBS Scientific, Del Mar, Calif.]) equipped with germicidal UV lamps. Other precautions to prevent carryover contamination of amplified DNA included different sets of pipettes dedicated for DNA extraction, PCR setup, and postamplification activities; the use of aerosol barrier filter pipette tips; and exposure of PCR tubes, pipettes, and tips to UV light prior to PCR setup. Each PCR included a negative control sample with sterile water as template and a positive control sample with B. andersonii (MOK-1C) culture extract.
Amplicons were visualized on 2% agarose gels stained with ethidium bromide and were documented with a digital gel imaging system (GelDocMega; BioSystematica, Devon, United Kingdom).
DNA purification and sequencing. PCR-amplified gene fragments were purified of primers and other nonspecific amplification by-products by using the QIAquick PCR purification kit (Qiagen) and were sequenced for species confirmation and phylogenetic comparison. Because of the large number of amplicons analyzed in this study, samples were sequenced in only one direction, using the nested forward primer for each target gene fragment. DNA templates were sequenced by using the fluorescent dideoxy terminator method of cycle sequencing on either a Perkin-Elmer Applied Biosystems (ABI) 373A or 377 automated DNA sequencer, according to ABI protocols (29). Sequences were generated by using Sequencher software (Gene Codes Corporation, Ann Arbor, Mich.).
Sequence analysis. Investigator-derived sequences were compared with those obtained by searching the GenBank database (National Center for Biotechnology Information) with the Basic Local Alignment Search Tool (1) and were aligned by using Clustal X (54). The GenBank accession numbers used for comparison with the B. lonestari flaB gene sequences reported in this study are AF264901, AF273670, AF298653, AF408410, D43777, D82859, D82861, D82862, D82863, D82864, D86618, U26704, U26705, U28498, U28499, X15661, X75202, and X75204. The accession numbers used for comparison with the B. burgdorferi sensu lato flaB gene sequences reported here are AB035595, AF264883, AF264886, AF264889, AF264892, AF264894, D82847, D82849, D82852, D82854, D82856, D82857, D83762, D83763, L29245, U26704, X16933, X75200, X75202, and X75203. The accession numbers used for comparison with the B. burgdorferi sensu lato p66 gene sequences are AE001161, AY090473, U96240, U96241, U96243, and X87727, and those used for comparison with the B. burgdorferi sensu lato ospA gene sequences are A24008, AB016975, AF186846, AF369944, AY030279, L23144, X80257, U20360, U65802, X16467, Y10838, Y10840, Y10892, Y10897, and Z29087. Phylogenetic trees were constructed by using the neighbor-joining (NJ) and unweighted pair-group method with arithmetic mean (UPGMA) distance methods and by parsimony analysis (46, 53). Tree topologies and evolutionary relationships obtained with the different methods were compared for consensus. The tree-building program was MEGA 2.1 (24). To estimate the node reliability of trees obtained with each method, bootstrap values (13) based on an analysis of 1,000 replicates were determined. Distance matrices were generated by the methods of Jukes and Cantor (21) and by the Kimura two-parameter model for multiple substitutions (22).
Nucleotide sequence accession numbers. The GenBank accession numbers for the B. lonestari flaB gene sequences reported in this study are AY654941 to AY654945, those for the B. burgdorferi sensu lato flaB gene sequences reported here are AY654901 to AY654918 and AY654946, those for the B. burgdorferi sensu lato p66 gene sequences are AY654926 to AY654940, and those for the B. burgdorferi sensu lato ospA gene sequences are AY654919 to AY654925.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES RESULTS Borrelia isolation. Early in the study, attempts were made to isolate Borrelia from vertebrate ear tissue samples from a small number of animals (n = 22) and from adult blacklegged ticks (n = 72) from UNFWS and GRSPWMA. The animals included 1 golden mouse, 18 cotton mice, and 3 rice rats. No viable spirochetes were observed in the vertebrate or tick specimen cultures, despite PCR amplification of various B. burgdorferi sensu lato target gene fragments from the ear tissue extracts from most of the vertebrates (see below) and from a few of the tick extracts and excellent growth of B. burgdorferi sensu lato reference strain cultures inoculated into aliquots of the same medium and maintained under identical conditions. Because of the lack of agreement between culture isolation results and PCR, no further attempts were made to isolate Borrelia in this study.
Borrelia species-specific PCR. Borrelia sp. flaB DNA that was determined to be from B. lonestari (see below) was detected in eight samples from host-seeking adult lone star ticks from four study sites, three in northeastern Florida and one in the north-central part of the state (Table 2 and Table 3; Fig. 1). A few of the positive samples were from ticks extracted and tested as a pool, and a few were from extracts from single ticks (Table 2). Each positive tick pool was treated as if it contained a single positive tick. All of the Borrelia sp. flaB PCR-positive lone star tick samples also tested positive with Borrelia sp.-specific 16S rDNA primers. The B. lonestari infection prevalence among ticks at individual sites ranged from 0 to 4.8%. The overall prevalence among 396 ticks was 2.0% (Table 3).
B. burgdorferi sensu lato-specific PCR. B. burgdorferi sensu lato flaB DNA was amplified from DNA extracts from seven small mammal and three tick species in Florida (Table 2, Table 4, and Table 5). Approximately 85% (56 of 66) of small vertebrates tested positive (Table 4), including two new hosts recorded for B. burgdorferi sensu lato: the southern flying squirrel (Glaucomys volans) and the golden mouse (Ochrotomys nuttalli). The infection prevalences were very similar among vertebrates at both sites. PCR-positive DNA extracts were obtained from vertebrate ear tissue and Nobuto blood samples. Thirty of 40 flaB-positive mammal ear tissue samples (75%) tested positive with the p66 assay also. However, only a smaller fraction (12 of 37; 32%) of flaB-positive samples also tested positive with either set of ospA or the 5S-23S ISR primers. These results were consistent upon retesting of many of the samples several times with each primer set. Experimentation with different amounts of starting template and modifications of PCR amplification parameters did not vary the results.
B. burgdorferi sensu lato flaB DNA was detected in five extracts from host-seeking adult lone star ticks from two sites (Table 2 and Table 5). One of the pooled samples (AA15POOL)tested positive for both B. lonestari and B. burgdorferi sensu stricto (Table 2; Fig. 2 and 4). The overall prevalence among all lone star ticks tested from four sites was 2.0% (Table 5). Because Ixodes affinis adults were collected in only small numbers at any given site, all ticks tested from 10 different sites were combined to estimate the infection prevalence in that species, which was nearly 31% (Table 5). Positive I. affinis ticks came from five sites in Florida and one in southeastern Georgia (Cumberland Island, Camden County). B. burgdorferi sensu lato flaB DNA was detected in adult I. scapularis from all 13 sites in northern Florida from which ticks were tested (Table 2; Fig. 1). Enough ticks were tested from two coastal sites to estimate the infection prevalence, which was 4.6% among 216 ticks (Table 5).
Borrelia species flagellin sequences. Florida B. lonestari flaB sequences, consisting of approximately 320 nucleotides of data, were more than 99% similar to all B. lonestari sequences in GenBank and clustered with other B. lonestari sequences in the phylogenetic trees produced by various tree-building methods (Fig. 2). All of the Florida sequences were identical to the sequence with accession number AF298653 from a lone star tick in Alabama (7) and to two sequences derived from a previously reported human patient and an attached lone star tick (accession numbers AF273670 and AF273671, respectively) (18). The sequence from the amplicon produced with the Borrelia sp.-specific flaB primers for the Florida AA4POOL sample was 99.1% similar to the sequence for B. burgdorferi sensu stricto B31 and clustered with it in the phylogenetic tree (Fig. 2). All other lone star tick samples that produced flaB sequences similar to those of the B. burgdorferi sensu lato complex were obtained with the B. burgdorferi sensu lato-specific PCR primers.
B. burgdorferi sensu lato flagellin, p66, and ospA sequences. Florida B. burgdorferi sensu lato flaB amplicons derived from several different small mammal and tick species extracts were sequenced, and the sequences were compared to B. burgdorferi sensu lato reference strain sequences. Approximately 362 nucleotides of data were compared. The phylogenetic trees created by different methods were very similar. All of the Florida flaB sequences clustered with reference strains of either B. burgdorferi sensu stricto or B. bissettii (Fig. 3). Sequences that clustered with B. burgdorferi sensu stricto reference strains were obtained from all vertebrate and tick species tested (Table 2; Fig. 3), including lone star ticks. A smaller number of Florida sequences clustered with B. bissettii reference strains. Sequences that clustered in this group were obtained from cotton mice, cotton rats, a rice rat, and I. affinis (not all shown in Fig. 3).
The phylogenetic trees constructed with p66 sequences also clustered Florida sequences with either B. burgdorferi sensu stricto or B. bissettii reference strains. Figure 4 shows the NJ tree obtained with p66 sequences. In this analysis, B. bissettii 25015 was located most closely on the tree to B. andersonii MOK-1c, rather than being clustered with other B. bissettii strains from Florida and Colorado. Another difference in comparison to the flaB trees was that the sequence for strain SCW-30 h, isolated from an I. minor tick in South Carolina (26), was not placed near B. burgdorferi sensu stricto strains. The NJ and UPGMA bootstrap consensus trees (not shown) and the maximum-parsimony tree (data not shown) also placed the SCW-30h p66 sequence on a separate branch from all other B. burgdorferi sensu lato strains included in the analysis. However, based on the flaB sequence analysis, this strain is most similar to B. burgdorferi sensu stricto and B. bissettii.
None of the Florida ospA sequences analyzed in this study represented strains of B. burgdorferi sensu stricto; all clustered with B. bissettii reference strains (Fig. 5). The phylogenies obtained with ospA sequences were very similar regardless of the tree construction method and generally are in agreement with those derived from analysis of flaB and p66 sequences. For example, based on ospA sequence data, B. bissettii 25015 clustered with other B. bissettii strains, and SCW-30h clustered with B. burgdorferi sensu stricto strains, albeit somewhat distantly (Fig. 5). The primary difference between the flaB and ospA phylogenies for strains of B. burgdorferi sensu lato analyzed in this study was that the B. andersonii ospA sequences were most similar to a those of a strain of Borrelia valaisiana (VS116), while B. andersonii flaB sequences were most similar to those of B. burgdorferi sensu stricto.
Top Abstract MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES DISCUSSION DNA amplification and sequence analysis showed that most of the B. burgdorferi sensu lato strains identified in this study from small mammals and ticks were very similar to B. burgdorferi sensu stricto, including strains identified in the animals and ticks whose tissues were cultured but from which no spirochetal isolates were recovered. ospA- and 5S-23S ISR-positive samples in this study clustered with reference strains of B. bissettii in the phylogenetic trees created with their flagellin, p66, and ospA sequences. Only the B. bissettii strains identified in this study amplified a product of the expected size and sequence with the ospA and 5S-23S ISR primers used. All of the flagellin and p66 sequences from ospA-negative samples clustered with B. burgdorferi sensu stricto strains. Probably all or nearly all of the other ospA-positive strains were also B. bissettii strains, and all or nearly all of the ospA-negative strains were most like B. burgdorferi sensu stricto. Based on this assumption, 79% (44 of 56) of the flagellin-positive small mammals were infected with B. burgdorferi sensu stricto, and 21% (12 of 56) were infected with B. bissettii. B. bissettii strains were identified in four vertebrate species: cotton rat (n = 8), golden mouse (n = 1), rice rat (n = 2), and cotton mouse (n = 1). Based on the amplicons sequenced in this study, the only tick species infected with B. bissettii was I. affinis. It is possible, however, that some PCR-positive I. scapularis ticks contained B. bissettii but that those amplicons were not sequenced. B. burgdorferi sensu stricto strains were found in all vertebrate species tested, as well as in host-seeking adult A. americanum, I. affinis, and I. scapularis.
The various ospA serotypes of B. burgdorferi sensu lato have been associated with different clinical manifestations of LD (58). Some B. burgdorferi sensu lato strains isolated in Europe (57) and several strains isolated from I. scapularis ticks removed from humans and vegetation in the northeastern United States (2) lacked ospA. However, most B. burgdorferi sensu stricto strains isolated in the United States express ospA, or some form of ospA or ospB, including those from the southeastern United States (36). The ospA sequences of distinct B. burgdorferi sensu lato species vary significantly, while those of most B. burgdorferi sensu stricto strains analyzed thus far are very homogeneous (28, 57).
In comparison, B. andersonii strains have shown significant heterogeneity in their reactivity to specific ospA monoclonal antibodies (35). Strain MOD-6, isolated from lone star tick larvae removed from a rabbit in Missouri, failed to react with ospA monoclonal antibodies H3TS and H5332, as well as monoclonal antibody H6831 for ospB, in a previous study (35). That strain also failed to amplify an ospA fragment with one of three different ospA PCR primer sets. Using the primers described in Table 1, it was not possible to amplify detectable ospA from any B. burgdorferi sensu stricto strain characterized in the present study. These southern B. burgdorferi sensu stricto strains may simply lack ospA, as do previously described strains from the northeastern United States (2). Another possible explanation is that ospA is expressed by these strains but the ospA genes of these strains differ significantly enough in the region of the primers used in the present study to prevent correct primer annealing or to reduce the sensitivity of amplification of the target gene fragments below the level needed to identify them via electrophoresis and UV transillumination, despite the use of highly sensitive nested PCR assays. Recombination between ospA and ospB proteins, resulting in the deletion of osp gene sequences and the creation of chimeric gene fusions, has been described for some B. burgdorferi sensu lato strains (45, 48).
The ospA-negative samples in the present study also failed to amplify a product in PCRs with the nested 5S-23S ISR PCR assay, suggesting the possibility of atypical differences in rRNA arrangement in these same strains. Such findings have been reported in other studies. For example, a B. bissettii 25015-like strain isolated from a patient in Slovenia failed to amplify a product with a different set of 5S-23S ISR primers (39). Two strains of B. andersonii (21038 and 19857) possess a single copy of 5S (rrfA) and an interrupted or fragmented second copy of 23S (rrlB), rather than the typical two complete copies of each, and a B. japonica strain (IKA2) contains only single copies of both 23S (rrlA) and 5S (rrfA) (27). The lack of a second copy of 23S (rrlB) prevented amplification of the IKA2 product with forward and reverse primers located within the two copies of 23S (rrlA and rrlB, respectively), but a product was amplified with primers located within the first copies of 23S and 5S (rrlA and rrfA) (27).
All of the B. bissettii strains identified in small mammals and ticks from Florida, the B. andersonii MOK-1c reference strain, and several B. burgdorferi sensu stricto reference strains amplified products of the expected size with the 5S-23S ISR and both ospA PCR assays used in this study, demonstrating the ability of the primers to amplify strains of genetically distinct species groups from many samples. Nevertheless, the ospA- and 5S-23S ISR-negative PCR results in this study could stem from inadequate sensitivity of those assays due to low copy numbers of the target genes in some experimental samples. Contamination of PCR samples does not explain the flaB- and p66-positive but ospA- and 5S-23S ISR-negative PCR results with B. burgdorferi sensu stricto strain samples from Florida, since B. andersonii MOK-1c was used as a positive control in the testing. Contamination of DNA extracts with reference strain culture sample DNA also cannot explain the findings, since none of the Florida B. burgdorferi sensu stricto flaB or p66 sequences were identical to those of the cultured strains.
The patterns of PCR positivity and negativity observed in this study suggest that the predominant strains of B. burgdorferi sensu lato in the study region may comprise a more genetically distinct group of B. burgdorferi sensu stricto than has previously been described. This group of B. burgdorferi sensu stricto strains may be resistant to culture in BSK-H medium and be variable in ospA and 5S-23S rRNA gene expression and/or arrangement. Alternatively, these strains may be cultivable, but perhaps they result in very low spirochetemia, below the level of detection via isolation in BSK-H medium and DNA amplification with the ospA and 5S-23S ISR primers used in this study. Additional testing of the ospA- and 5S-23S ISR-negative samples from Florida with other primers may show whether ospA and rRNA variation similar to that for some B. andersonii or B. japonica strains explains the PCR results in the present study. If so, it will be interesting to learn whether such strains also exist elsewhere. Previous studies that relied on culture isolation, ospA PCR assays, or 5S-23S ISR PCR testing for initial detection of B. burgdorferi sensu lato could have failed to recognize such strains present in the respective areas, just as the present study would have failed to identify them had it relied solely upon similar methods of detection. Even more intriguing are the potential human disease implications if such strains are capable of infecting humans and causing Lyme disease-like manifestations. Would human blood, tissues, or other specimens from patients infected with similar strains and tested with typical diagnostic tests, including antibody tests, culture, or PCR, produce positive results? Alternatively, these strains may not be infectious or pathogenic to humans.
Many spirochete species (e.g., Treponema pallidum) have proven difficult to cultivate. Previous efforts to isolate Borrelia spp. from lone star ticks in BSK medium failed. B. lonestari was identified and described based solely on DNA amplification and sequence analysis and was only recently isolated in a tick cell line (55). It has been demonstrated that isolation in BSK medium does not detect all genotypes of B. burgdorferi sensu lato circulating in a given area. The genetic diversity of B. burgdorferi sensu lato detected in samples from humans, other vertebrates, and ticks via PCR amplification is greater than that detected by initial culture of spirochetes in BSK (30, 32). Even DNA testing via PCR amplification for detection, if based on amplifying some genes that vary considerably such as ospA, may not be adequately sensitive or reliable for detecting all B. burgdorferi sensu lato strains in an area.
The results of this study suggest that future studies aimed at identifying the full diversity of B. burgdorferi sensu lato strains in a given area should use highly sensitive, DNA amplification-based methods that target conserved genes. The nested B. burgdorferi sensu lato flaB PCR assay used in this study proved to be most reliable and identified strains present in ticks and small mammals that would not have been identified by the p66 or other assays. Moreover, the flaB-based phylogeny in the present study and results of other flagellin-based typing systems (15, 38) have agreed very well with other B. burgdorferi sensu lato typing methods, including sequence analysis of different gene targets, PCR-based restriction fragment length polymorphism analysis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis (56).
The B. lonestari infection prevalence (2%) in lone star ticks in Florida is similar to that found in other states (5, 52). If B. lonestari strains in A. americanum in Florida are pathogenic to humans, then the risk for STARI (Master's disease) is present in the study area. Considering the feeding habits and regional abundance of lone star ticks, this could explain a significant portion of the cases of Lyme disease-like illness recognized in Florida. However, the discovery of B. burgdorferi sensu stricto strains in an equal proportion of lone star ticks, along with the genetic heterogeneity identified in the strains in Florida that belong to this group, contribute to ongoing suspicions of this tick's involvement in transmitting B. burgdorferi sensu lato to humans. Although they are much less aggressive in biting humans in this region, I. scapularis ticks are also infected with B. burgdorferi sensu lato strains and could occasionally transmit them to people. The prevalence of infection in adult I. affinis ticks in Florida based on PCR testing (31%) was similar to that found in South Carolina (25.7%) by using culture isolation (11). However, this tick species is not known to bite humans and probably is important only in the enzootic transmission of the spirochete among small mammals, which are the preferred hosts for the immature tick stages (10).
This study showed via PCR testing that 4.6% of adult I. scapularis ticks from two coastal sites in Florida contained B. burgdorferi sensu lato DNA. Only 1.3% in South Carolina were infected, based on BSK culture results (11). The present study also identified a very high B. burgdorferi sensu lato infection prevalence (85%) among small mammals from two sites near the Atlantic Coast in northeast Florida. This is higher than the rates determined for small mammals in South Carolina, Georgia, and Florida in previous studies (11, 37). Interestingly, if infection prevalence data from a previous study (37) for cotton mice, cotton rats, and wood rats, three established small mammal reservoir species in the Southeast, are combined from all sites within those three states and compared, a potential trend is apparent. The combined prevalence measures among animals of the three species tested from South Carolina, Georgia, and Florida are 41.8% (82 of 196), 12.0% (33 of 274), and 6.5% (13 of 200), respectively (37). Those data were based upon initial isolation of spirochetes in BSK. The numbers of each species tested from each state were not equal, and the samples were collected from different sites within each state. Therefore, sampling bias could explain some of the variation. The higher vertebrate infection prevalence found in the present study compared to that for animals from Florida tested in the other study is most likely explained by my use of DNA amplification methods instead of culture isolation for detection of B. burgdorferi sensu lato. Taken together, these findings may be indicative of an actual north-south trend in the proportion of B. burgdorferi sensu lato strains that are cultivable in BSK rather than a trend in actual vertebrate infection prevalence. This supports a theory that the predominant B. burgdorferi sensu stricto strains in Florida (and perhaps in other southern states) may be difficult or impossible to cultivate in BSK. Comparisons of culture isolation and flaB PCR testing of samples from areas throughout the southeastern United States are needed to test this hypothesis.
Based on amplification and analysis of flagellin and p66 gene fragments, the predominant strains of B. burgdorferi sensu lato identified in this study in Florida are B. burgdorferi sensu stricto. However, they may be quite different from reference strains of B. burgdorferi sensu stricto in their ospA and 5S-23S rRNA genes. It is not known whether the Florida strains are pathogenic to humans or, even if they are, whether they are transmitted to humans. If these strains are pathogenic but can be transmitted to humans only by I. scapularis, then probably only low numbers of humans in this area become exposed or infected each year. The nymphal and adult stages of this tick species do not frequently parasitize humans in this region (14).
The lone star tick, however, is extremely aggressive and at all life stages bites humans. B. lonestari is a suspected human pathogen, and it was found in ticks at several sites in Florida. Yet, several pools of host-seeking adult lone star ticks were also infected with B. burgdorferi sensu stricto strains that are identical in their flaB and p66 sequences to the B. burgdorferi sensu stricto strains found in I. scapularis and small mammals in Florida. This may appear to disagree with the published findings that lone star ticks cannot acquire or maintain B. burgdorferi sensu lato This longstanding belief is based upon laboratory transmission studies conducted necessarily with reference strains of B. burgdorferi sensu stricto that were cultured or cultivable in BSK (40, 47). The B. burgdorferi sensu stricto strains described in this study are probably not identical to those, as described above, and evidence from this study suggests that they may not be as easily cultured, if cultivable at all, in BSK.
The findings of the present study extend the known geographic distributions of B. lonestari in A. americanum and of B. burgdorferi sensu lato in A. americanum, I. affinis, I. scapularis, and small mammals to new sites in northern and central Florida. They document new hosts for B. burgdorferi sensu lato infection, the flying squirrel and golden mouse, both of which may serve as additional reservoirs for the bacteria in the study area. The presence of B. burgdorferi sensu stricto strains in host-seeking lone star ticks at two sites in Florida suggests that A. americanum should still be considered a possible vector of at least some B. burgdorferi sensu lato strains. The implications of this study's findings for the human risk of infection in the southeastern United States with previously uncharacterized B. burgdorferi sensu lato strains or species or those not presently considered to be human pathogens, as well as the possibility of Lyme disease spirochete transmission from lone star ticks in addition to I. scapularis, demonstrate the need for further investigation of the ecology and epidemiology of borreliosis in the southern United States.
Acknowledgments I thank J. F. Piesman and B. S. Schneider for some Borrelia spirochete strains and for DNA amplification and sequence confirmation for some strains analyzed early in the study. I thank J. H. Oliver, Jr., for also providing Borrelia spirochete strains. I am grateful to A. J. Hendricks, J. Manns, B. Maton, and K. Overly for assistance in field and lab work associated with this project.
This work was supported in part by a research grant from the American Lyme Disease Foundation, Somers, N.Y., and a University of North Florida Dean's Research Professorship funded by the Brooks Health Foundation, Jacksonville, Fla.
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An ospA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and ospA sequence analysis. J. Clin. Microbiol. 31:340-350. [Free Full text in PMC]. Figures and Tables FIG. 1. Map of Florida showing geographic distribution of Borrelia species detected in ticks via nested flagellin gene PCR. Circles indicate locations of study sites with B. burgdorferi sensu lato-positive blacklegged ticks, I. affinis, or lone star ticks. Triangles (more ...) FIG. 2. Unrooted neighbor-joining phylogenetic tree based on a comparison of partial flagellin gene sequences obtained from Florida lone star ticks with other Borrelia species. B. burgdorferi sensu stricto strain B31 was included as an outgroup. Numbers at the (more ...) FIG. 3. Unrooted UPGMA phylogenetic tree based on a comparison of partial flagellin gene sequences obtained from Florida small mammals and ticks with other B. burgdorferi sensu lato species. B. lonestari was included as an outgroup. Numbers at the branch nodes (more ...) FIG. 4. Unrooted neighbor-joining phylogenetic tree based on a comparison of partial p66 gene sequences obtained from Florida small mammals and ticks with other B. burgdorferi sensu lato species. Numbers at the branch nodes represent bootstrap values as percentages (more ...) FIG. 5. Unrooted neighbor-joining phylogenetic tree based on a comparison of partial ospA gene sequences obtained from Florida small mammals and ticks with other B. burgdorferi sensu lato species. Numbers at the branch nodes represent bootstrap values as percentages (more ...) TABLE 1. Oligonucleotide primers used in this study TABLE 2. Borrelia strains identified in the present study TABLE 3. Prevalence of B. lonestari flagellin DNA among lone star ticks collected in Florida, 1999 to 2000 TABLE 4. Prevalence of B. burgdorferi sensu lato flagellin DNA among small mammals collected in Florida, 1999 to 2000 TABLE 5. Prevalence of B. burgdorferi sensu lato flagellin DNA among host-seeking adult ticks collected in northern Florida and southeastern Georgia, 1999 to 2000
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