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Borrelia lusitaniae and green lizards (Lacerta viridis), Karst Region, Slovakia.



In Europe, spirochetes within the Borrelia burgdorferi Borrelia burg·dor·fe·ri
n.
A spirochete causing Lyme disease in humans.


Borrelia burgdorferi The spirochete agent of Lyme disease, which contains several outer membrane proteins and a highly immunogenic flagellar
 sensu lato complex are transmitted by Ixodes ricinus ticks. Specific associations are described between reservoir hosts and individual genospecies. We focused on green lizard (Lacerta viridis) as a host for ticks and potential host for borreliae. In 2004 and 2005, a total of 146 green lizards infested in·fest  
tr.v. in·fest·ed, in·fest·ing, in·fests
1. To inhabit or overrun in numbers or quantities large enough to be harmful, threatening, or obnoxious:
 by ticks were captured, and 469 I. ricinus ticks were removed. Borrelial infection was detected in 16.6% of ticks from lizards. Of 102 skin biopsy Skin Biopsy Definition

A skin biopsy is a procedure in which a small piece of living skin is removed from the body for examination, usually under a microscope, to establish a precise diagnosis.
 specimens collected from lizards, 18.6% tested positive. The most frequently detected genospecies was B. lusitaniae (77.9%-94.7%). More than 19% of questing I. ricinus collected in areas where lizards were sampled tested positive for borreliae. B. garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae.

**********

The causative agents of Lyme borreliosis Lyme borreliosis
Another name for Lyme disease.

Mentioned in: Lyme Disease
, spirochetes of the Borrelia burgdorferi sensu lato complex, are maintained in natural foci by circulation between the vector ticks in the Ixodes ricinus complex and reservoir hosts of various vertebrate taxa taxa: see taxon. . The B. burgdorferi s.l. complex encompasses 12 species (1-3); 4 species have been clearly established as pathogenic to humans: B. afzelii, B. garinii, B. burgdorferi s. s., and B. spielmanii (4-6). B. valaisiana and B. lusitaniae, which were previously considered nonpathogenic, may cause disease as well (7,8). Different species are associated with distinct ecologic features, levels of pathogenicity, and clinical symptoms in patients.

In Europe, I. ricinus ticks infest in·fest
v.
1. To live as a parasite in or on tissues or organs or on the skin and its appendages.

2. To inhabit or overrun in numbers large enough to be harmful, threatening, or obnoxious.
 a wide variety of vertebrate hosts, such as mammals, birds, and lizards. The vertebrate hosts are necessary to maintain the tick population and may also serve as reservoirs for the pathogen. Therefore, the identification of reservoir host species is essential to clarify the transmission patterns of B. burgdorferi s.l. in natural foci. The importance of rodents for maintaining B. afzelii (9), and of birds for B. garinii and B. valaisiana (10), in endemic regions of Slovakia is now indisputable. The National Park Slovak Karst The Slovak Karst (Slovak: Slovenský kras) is one of the mountain ranges of the Slovenské rudohorie Mountains in the Carpathians in southern Slovakia. It consists of a complex of huge karst plains and plateaus.  is within the region in which B. burgdorferi s.l. in questing ticks and birds has been reported (V. Taragel'ova, unpub. data). In this area, 2 lizard species occur sympatrically, the common wall lizard (Zool.) a common European lizard (Lacerta muralis) which frequents houses, and lives in the chinks and crevices of walls; - called also wall newt.

See also: Wall
 (Podarcis muralis) and the green lizard (Lacerta viridis). The green lizard, the Lizard, The, peninsula, Cornwall, SW England. Its southern extremity (the southernmost point of Great Britain) is called Lizard Point or Lizard Head. The coast has colored serpentine rocks, small coves and bays, wave-hollowed caves, islets (e.g.  dominant species, is frequently infested by immature stages of I. ricinus ticks (11).

The importance of lizards in the maintenance cycles of B. burgdorferi s.l. spirochetes is still controversial. In Italy, B. lusitaniae was detected in blood and tissue samples of P. muralis (12). Furthermore, Psammodromus algirus, the most abundant lizard species in North Tunisia, was found to be the primary host for immature stages of I. ricinus. Thus, it could play a role in the circulation of borreliae (13). B. burgdorferi s.s., B. andersonii, and B. bisettii were detected in the blood of 9 lizard species in the southeastern United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area.  (14). On the other hand, several other studies in the United States have shown that the lizards Sceloporus occidentalis Noun 1. Sceloporus occidentalis - common western lizard; seen on logs or rocks
blue-belly, western fence lizard, swift

fence lizard - spiny lizard often seen basking on fences in the United States and northern Mexico
 and Elgaria multicarinata are reservoir-incompetent for borreliae because they possess borreliacidal factor in their blood (15,16). However, 2 lizard species, Eumeces inexpectatus and Anolis carolinensis, can sustain B. burgdorferi s. s. infection (17).

In the Slovak Karst (southeastern part of Slovakia), the green lizard is the major host for immature stages of I. ricinus ticks (11). Therefore, the main aim of this study was to find out whether green lizards can participate in the maintenance cycles of B. burgdorferi s.l. in natural foci and whether an association with specific borrelial genospecies exists.

Materials and Methods

Study Area

The study was conducted in the National Park Slovak Karst. This area represents a part of the Inner Carpathians in southeastern Slovakia (48 [degrees] 36'N, 20 [degrees] 52'E). The climate is warm with low humidity and average temperatures of 4 [degrees] C in January and 18 [degrees]C in July. The average rainfall is 700 mm/year.

Tick and Lizard Collection

This survey was conducted in 2004-2005, from May to September, when lizards and ticks are active. Questing I. ricinus nymphs and adults were collected by flagging the vegetation in areas where lizards were sampled. Ticks were immediately stored in 70% ethanol.

Green lizards were captured along hiking paths by hand or by noosing, in which a loop made from fishing nylon was attached to the end of a wooden stick and dangled in front of a lizard, which would be captured as it walked through the loop. Animals were characterized by sex and age (adult, subadult, juvenile) and examined for ticks.

Ticks were removed with forceps immediately after capture and stored in 70% ethanol. Biopsy specimens (a 2-cm distal part of the tail and a 1-mm x 1.5-mm piece of skin from collar scales) were taken from each lizard with sterile scissors scissors

Cutting instrument or tool consisting of a pair of opposed metal blades that meet and cut when the handles at their ends are brought together. Modern scissors are of two types: the more usual pivoted blades have a rivet or screw connection between the cutting ends
 and put in separate vials with 70% ethanol. Ticks were identified to the species and sex. Only I. ricinus ticks were further examined for B. burgdorferi sensu lato.

DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 Isolation

Immediately before extraction, ticks and tissues were dried for 30 min to evaporate the ethanol. Each sample was cut with a disposable sterile scalpel. Tissue DNA from lizards' tails and scales was extracted by using DNeasy tissue kit (Qiagen, Hilden, Germany). Extraction steps were conducted according to according to
prep.
1. As stated or indicated by; on the authority of: according to historians.

2. In keeping with: according to instructions.

3.
 the manufacturer's protocol. Genomic DNA genomic DNA
n.
The full complement of DNA contained in the genome of a cell or organism.
 from ticks was isolated by alkaline hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds.  (18). Incubation time was extended from 5 to 30 rain. Isolated DNA was stored at -20[degrees]C.

PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 


PCR amplification was performed in a 25-[micro]L reaction mixture from the MasterTaq DNA polymerase DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template.  kit (Eppendorf AG, Hamburg, Germany) containing 10.4 [micro]L deionized water Deionized water (DI water or de-ionized water; also spelled deionised water, see spelling differences) is water that lacks ions, such as cations from sodium, calcium, iron, copper and anions such as chloride and bromide. , 5 [micro]L 5x TaqMaster PCR Enhancer, 2.5 [micro]L 10x Taq buffer (with 15 mmol/L [Mg.sup.2+]), 1.5 [micro]L 25-mmol/L solution of Mg [(OAc).sub.2], 0.1 [micro]L Taq DNA polymerase (5 U/[micro]L), 0.5 [micro]L deoxynucleoside triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.

tri·phos·phate
n.
A salt or ester containing three phosphate groups.
 (dNTP) mix (10 mmol/L) (Fermentas, Vilnius, Lithuania), 1.25 [micro]L of each primer (10 pmol/[micro]L) (Invitrogen, Paisley, Scotland), and 2.5 [micro]L DNA template.

To verify that DNA had been successfully isolated from each tick, primers for the fragment of the tick's mitochondrial mitochondrial

pertaining to mitochondria.


mitochondrial RNAs
a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by a mitochondrial-specific RNA polymerase, that account for about 4% of the total cell RNA that
 cytochrome cytochrome (sī`təkrōm'), protein containing heme (see coenzyme) that participates in the phase of biochemical respiration called oxidative phosphorylation.  b gene (620 bp) were used (19). Negative samples were excluded from the further analysis. Positive samples were examined for the presence of B. burgdorferi s. l. by amplifying a portion of the 5S (rrfA)-23S (rrlB) rDNA intergenic spacer (20). PCR products were subjected to electrophoresis on a 1% agarose agarose

more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments.
 gel, stained with ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic acid stain in molecular biology laboratories for techniques such as agarose gel electrophoresis. , and visualized with a UV transilluminator.

RFLP RFLP
abbr.
restriction fragment length polymorphism



RFLP

restriction fragment length polymorphism.

RFLP 
 Analysis

The positive PCR products of the 5S-23S rDNA intergenic spacer regions were further analyzed by restriction fragment length polymorphism restriction fragment length polymorphism
n. Abbr. RFLP
Intraspecies variations in the length of DNA fragments generated by the action of restriction enzymes and caused by mutations that alter the sites at which these enzymes act, changing
 (RFLP). Previously extracted DNA of B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi s.s. were used as positive controls. For each positive sample, 13 [micro]L amplified DNA was digested at 65 [degrees] C overnight in a solution containing 5 U of TrulI (300 U/mL) and 1 x Buffer R (Fermentas). Electrophoresis was conducted in 16% polyacrylamide gel pol·y·a·cryl·a·mide gel
n.
A hydrated polymer consisting of a long chain of amide groups, used as a medium for substances that undergo gel electrophoresis.
 at 150 V for 3 h. The gels were stained with SYBR SYBR Synergy Brands, Inc. (stock symbol)  gold nucleic acid nucleic acid, any of a group of organic substances found in the chromosomes of living cells and viruses that play a central role in the storage and replication of hereditary information and in the expression of this information through protein synthesis.  gel stain (Molecular Probes Molecular Probes is a biotechnology company located in Eugene, Oregon specializing in fluorescence. The company was founded in 1975 by Richard and Rosaria Haugland in their kitchen in Minnesota, then moved briefly to Texas and finally to Oregon in the early 1980s. , Leiden, the Netherlands) for 20 min, and bands were visualized with a UV transilluminator. RFLP profiles that differed from the known profiles of positive controls were further analyzed by sequence analysis.

DNA Sequencing DNA sequencing

The determination of the sequence of nucleotides in a sample of DNA.
 of PCR Products

Sequencing was performed at the Department of Molecular Biology molecular biology, scientific study of the molecular basis of life processes, including cellular respiration, excretion, and reproduction. The term molecular biology was coined in 1938 by Warren Weaver, then director of the natural sciences program at the Rockefeller  (Faculty of Natural Sciences Commenius University, Bratislava, Slovak Republic). PCR of the 5S-23S rDNA intergenic spacer was conducted according to the protocol described above. For the fla gene amplicons, DNA strands were sequenced as described previously (21). PCR products were purified by using a QIAquick PCR purification kit (Qiagen). The complementary strands of each sequenced product were manually assembled. Sequences were compared with GenBank entries by Blast N2.2.13 (22). Homologous homologous /ho·mol·o·gous/ (ho-mol´ah-gus)
1. corresponding in structure, position, origin, etc.

2. allogeneic.


ho·mol·o·gous
adj.
1.
 sequences were aligned by using the CLUSTAL W Multiple Sequence Alignment A multiple sequence alignment (MSA) is a sequence alignment of three or more biological sequences, generally protein, DNA, or RNA. In general, the input set of query sequences are assumed to have an evolutionary relationship by which they share a lineage and are descended from a  Program (version 1.81) (23). Sequence similarity among the sequences were calculated by EMBOSS em·boss  
tr.v. em·bossed, em·boss·ing, em·boss·es
1. To mold or carve in relief: emboss a design on a coin.

2.
 Align, a pairwise alignment algorithm (http://www.ebi.ac.uk/emboss/align).

The accession numbers of 5S-23S rDNA intergenic spacer sequences obtained in this study are DQ539339 and DQ539340. Accession numbers of flagellin flagellin /fla·gel·lin/ (flah-jel´in) a protein of bacterial flagella; it is composed of subunits in several-stranded helical arrangement.  sequences obtained in this study are DQ788618, DQ788619, and DQ788620.

Data Analysis and Statistics

To estimate the probability of a tick's becoming infected after engorging on a green lizard and to measure the degree of infectiousness of infected animals, specific infectivity [I.sub.s] (24) and transmission coeficient [[beta].sub.H-T] (9) were calculated. Individual infectivity (i) is defined as the proportion of larvae Larvae, in Roman religion
Larvae: see lemures.
 derived from an individual lizard that are infected (i = [l.sub.i]/[l.sub.h], [l.sub.i] is the number of larvae that become infected, and [l.sub.h] is the total number of larvae derived from that host). The specific infectivity ([I.sub.s]) of a reservoir host species is defined as the sum of individual infectivities and number of individual lizards sampled ([I.sub.s] = [summation][i.sub.S]/[n.sub.S], n is the number of individual ticks captured). The host-to-tick transmission coefficient Transmission coefficient could refer to:
  • Transmission coefficient (optics), used to represent optical transmission
  • Transmission coefficient (chemistry), used in the Arrhenius equation
  • Transmission coefficient (physics), used in quantum mechanical tunnelling
 ([[beta].sub.H-T]) is defined as the portion of the sum of individual infectivities and the number of lizards that infected [greater than or equal to] 1 larva larva, in zoology
larva, independent, immature animal that undergoes a profound change, or metamorphosis, to assume the typical adult form. Larvae occur in almost all of the animal phyla; because most are tiny or microscopic, they are rarely seen.
 ([[beta].sub.H-T] = [summation][i.sub.S]/[n.sub.iS] ([n.sub.i] is the number of individual hosts that gave rise to at least 1 infected tick). Differences in the prevalence of B. burgdorferi s.l. in I. ricinus were evaluated statistically with the 2-tailed [chi square chi square (kī),
n a nonparametric statistic used with discrete data in the form of frequency count (nominal data) or percentages or proportions that can be reduced to frequencies.
] test (degrees of freedom [df] = 1). A value of p [less than or equal to] 0.05 was considered statistically significant.

Results

Lizards and Infestation infestation /in·fes·ta·tion/ (-fes-ta´shun) parasitic attack or subsistence on the skin and/or its appendages, as by insects, mites, or ticks; sometimes used to denote parasitic invasion of the organs and tissues, as by helminths.  with Ticks

One hundred forty-six (84 male, 52 female, and 10 subadult) of 165 (89 male, 61 female, and 15 subadult) captured green lizards were infested by ticks during the study period. In total, 469 (199 larvae and 270 nymphs) ticks were removed and further identified as I. ricinus. Male lizards were infested with 363 ticks (131 larvae and 232 nymphs), which represented 77.4% of all collected ticks. Moreover, 53 tails and 102 skin biopsy specimens were taken from the captured lizards.

B. burgdorferi Prevalence in Ticks Collected from Lizards

DNA isolation was successful in 464 ticks (197 larvae and 267 nymphs), from which the fragment of cytochrome b gene was amplified. These ticks were further analyzed for the presence of B. burgdorferi s.l. In total, 77 (16.6%) ticks carried borreliae. The infection prevalence between nymphs (15.2%) and larvae (17.6%) did not differ significantly (p = 0.49669, df = 1) (Table 1). Twenty-nine percent of tick-infested lizards carried [greater than or equal to] 1 infected tick. Infected lizards yielded [approximately equal to] 2 infected larvae per host.

Genotyping with PCR-RFLP PCR-RFLP Polymerase Chain Reaction–Restriction Fragment Length Polymorphism  identified the following species: B. lusitaniae, B. afzelii, B. garinii, B. burgdorferi s.s., and B. valaisiana. Of the B. burgdorferi-positive ticks, most (77.9%) were infected with B. lusitaniae. The presence of this species was significantly higher than that of other species (p [less than or equal to] 0.001). B. lusitaniae was detected in 26 (86.7%) larvae. B. afzelii, B. garinii, and B. burgdorferi s. s. each were found in 1 larva. Of the 47 B. burgdorferi-infected nymphs, 34 (72.3%) were infected with B. lusitaniae, 5 (10.6%) with B. afzelii, 2 (4.3%) with B. burgdorferi s. s., and 1 (2.1%) with B. garinii. A mixed infection of B. lusitaniae and B. burgdorferi sensu stricto was found in 5 (10.6%) nymphs (Table 1). Nymphs and larvae did not differ significantly in the prevalence of B. lusitaniae.

Male lizards were parasitized by 61 (79.2%) of 77 infected ticks. Variability of detected genospecies was higher in ticks collected from male than from female lizards. Larvae that fed on female lizards were only infected with B. lusitaniae. Out of 7 infected nymphs collected from females, B. lusitaniae was present in 5 and B. afzelii in 1 tick; B. lusitaniae and B. burgdorferi s.s. were detected as mixed infection in 1 nymph nymph, in Greek mythology
nymph (nĭmf), in Greek mythology, female divinity associated with various natural objects. It is uncertain whether they were immortal or merely long-lived. There was an infinite variety of nymphs.
. The specific infectivity from lizards to larval larval

1. pertaining to larvae.

2. larvate.


larval migrans
see cutaneous and visceral larva migrans.
 ticks was highest for B. lusitaniae. The specific infectivity of female lizards was slightly higher than that of males (Table 2).

B. burgdorferi Prevalence in Lizards

Isolated genomic DNA from tails and skin biopsy specimens from collar scales was tested for the presence of B. burgdorferi sensu lato. None of 53 tested tail samples was positive. Of 102 skin biopsy specimens collected from green lizards, 19 (18.6%) tested positive. Differences in infection prevalence between sexes (18.2% in males vs. 23.7% in females) were not significant. Of 9 skin biopsy specimens from subadult individual lizards, 2 (22.2%) were borrelia Borrelia

A genus of spirochetes that have a unique genome composed of a linear chromosome and numerous linear and circular plasmids. Borreliae are motile, helical organisms with 4–30 uneven, irregular coils, and are 5–25 micrometers long and 0.
 positive. The most frequently detected genospecies was B. lusitaniae (94.7%), which was present in 18 samples. One lizard was infected with B. afzelii.

B. burgdorferi Prevalence in Questing Ticks

Cytochrome b was amplified in 325 of 331 (71 female, 73 male, and 187 nymph) questing ticks. Therefore, only these 325 ticks (71 female, 71 male, and 183 nymph) were analyzed further for the presence of B. burgdorferi s.l. Sixty-three (19.3%) ticks tested positive. B. burgdorferi prevalence in female ticks was the same as in male ticks (22.5%), and it was lower in nymphs (16.9%).

RFLP analysis of the amplified products resulted in 5 distinct profiles. Of the 63 positive ticks, 21 (33.3%) were infected with B. garinii, 19 (30.2%) were infected with B. afzelii, 8 (12.7%) were infected with B. burgdorferi s. s., 7 (11.1%) were infected with B. lusitaniae, and 7 (11.1%) were infected with B. valaisiana. One nymph was infected simultaneously with B. garinii and B. valaisiana (Table 3).

Sequence Analysis

Representative samples of RFLP profiles that were different from the known profiles of positive controls were sequenced. The fragment of the 5S-23S rDNA intergenic spacer obtained from the B. burgdorferi s.l.-positive nymph (538N) from lizard belonged to B. lusitaniae. It was 100% identical to a Borrelia-positive skin biopsy specimen (277S) sampled from a lizard. Both obtained sequences were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB 091802) and 98.9%, 98.4%, and 94.5% similar to PotiBL37 (AY 463167), PotiB2 (L30131), and PotiB3 (L30132) strains from Portugal, respectively. To better characterize B. lusitaniae circulating in ticks and lizards from Slovak Karst, the fla gene from a B. burgdorferi s. k-positive questing adult tick and skin biopsy specimen from collar scale was amplified and sequenced. The flagellin sequence of B. lusitaniae detected in a skin biopsy specimen (277 S) was 100% identical and 99.6% similar to B. lusitaniae detected in questing adult ticks (43 ZLIF, 47 ZMLIM), respectively. Genotypes 277S and 43ZLIF were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB091812) as well as with the Polish strain D23-04 (DQ 016623). Genotype 47ZLIM was 99.6%, 99.6%, and 99.4% similar to Tr213, D23-04, and PotiB2 (DQ111036), respectively.

Discussion

The role of lizard species in maintaining B. burgdorferi s.l. has not been clearly elucidated yet. In United States, some lizard species have sustained borrelial infection (14,17); however, other species are incompetent reservoir hosts (15,16). The reservoir competence of lizards seems to be species specific. Therefore the aim of our study was to establish whether a relationship exists between green lizards, the dominant lizard species in the Slovak Karst, and B. burgdorferi s.l., which circulates in this area.

Seventeen percent of ticks that fed on lizards were infected with B. burgdorferi s.l. Seventy-eight percent of all infected ticks were infected with B. lusitaniae. Moreover, 18.6% of skin biopsy specimens from lizards were positive for B. burgdorferi s.l., and almost all (94.7%) were infected with B. lusitaniae. Similarly, B. lusitaniae have been detected in blood and tissue samples of Podarcis muralis in Tuscany in Italy, where borreliae were detected in 2 of 14 tested whole tails from lizards (12). At the beginning of our study, we also collected the distal tip of a lizard's tail because this method is minimally invasive and convenient for obtaining a tissue sample. The tissue at the tail, however, is squamous squamous /squa·mous/ (skwah´mus) scaly or platelike.

squa·mous or squa·mose
adj.
1. Covered with or formed of scales; scaly.

2.
 and keratinized, and none of the collected samples was borreliae positive. Therefore, we also obtained skin biopsy specimens from collar scales. These are elongated e·lon·gate  
tr. & intr.v. e·lon·gat·ed, e·lon·gat·ing, e·lon·gates
To make or grow longer.

adj. or elongated
1. Made longer; extended.

2. Having more length than width; slender.
 and extend from the skin on the ventral ventral /ven·tral/ (ven´tral)
1. pertaining to the abdomen or to any venter.

2. directed toward or situated on the belly surface; opposite of dorsal.


ven·tral
adj.
 side, so collecting them is minimally invasive and perhaps more likely to detect infection with B. burgdorferi s.l. because most of the immature L ricinus ticks parasitize par·a·sit·ize
v.
To live on or in a host as a parasite.



parasitize

to live on or within a host as a parasite.
 at the dorsal area (pers. observation). Furthermore, collar scales were chosen to avoid detecting the borreliae that persist in the skin after feeding of the infected ticks, which may enable infection of ticks by "extended co-feeding" (25). In this manner, incompetent host species may contribute to the circulation of B. burgdorferi s.l. in nature. For example, in England, I. ricinus ticks cofeeding on sheep become infected with B. burgdorferi, although sheep themselves are refractory to infection (26). In Europe the principal importance of cofeeding to Lyme disease Lyme disease, a nonfatal bacterial infection that causes symptoms ranging from fever and headache to a painful swelling of the joints. The first American case of Lyme's characteristic rash was documented in 1970 and the disease was first identified in a cluster at  ecology has been suggested to be the extent of the range of vertebrate host species that contribute significantly to the maintenance of B, burgdorferi s.l. spirochetes in nature (27). Therefore, cofeeding transmission could also be responsible for B. afzelii, B. garinii, and B. burgdorferi s.s. infection in larvae that fed on lizards collected in our study, even though skin biopsy results yielded mostly B. lusitaniae. Another possible explanation for the presence of non-B. lusitaniae spirochetes is that these larvae may have been infected transovarially (28). Cofeeding transmission might explain why individual lizards with borreliae negative skin biopsy specimens carry borreliae-positive larvae. Because the quantity of borreliae is low in the vertebrate host and may lodge in deeper organs, detecting them in skin biopsy specimens may not always be possible (29). Thus, a negative skin biopsy result does not prove conclusively that the lizard is not infected.

Despite the fact that male lizards hosted >75% of all host-feeding ticks, as well as 79.2% of all infected ticks, the specific infectivity and host-to-tick transmission coefficient were almost the same for male and female lizards. The seasonal activity of green lizards and different patterns in male and female behavior were monitored in the Slovak Karst (I. Majlath, unpub. data). Larger numbers of ticks feeding on male lizards are associated with higher male activity in spring months, when tick activity peaks as well. Male lizards end hibernation first and are active when the air temperature reaches 10 [degrees] C-12 [degrees] C. They need to restock re·stock  
tr.v. re·stocked, re·stock·ing, re·stocks
To furnish new stock for; stock again.

Verb 1. restock - stock again; "He restocked his land with pheasants"
 the energy that was depleted de·plete  
tr.v. de·plet·ed, de·plet·ing, de·pletes
To decrease the fullness of; use up or empty out.



[Latin d
 during winter and to gain energy for fighting other male lizards to compete for territory and females, for seeking female lizards, and for mating. Female activity increases in summer months when they are incubating eggs.

As determined by PCR, the overall prevalence of infection in our sample of questing ticks (19.3%) is consistent with 20.5% found in southern Czech Republic (20) but lower than that reported for a geographically close area in western Slovakia (40%-49%) (30). The total prevalence was higher in adults (22.5%) than in nymphs (19.6%), which is in agreement with the general pattern of increasing Borrelia prevalence through the life stages of ticks as their adults feed on a multiple hosts (31). The total prevalence of borreliae in male and female ticks was identical, but the distribution of genospecies was different. B. garinii was the predominant genospecies in this locality. B. garinii and B. valaisiana are the most commonly reported species in central Europe (32).

The high prevalence of B. lusitaniae in borrelia-positive larvae and nymphs as well as skin biopsy specimens from lizards suggests that green lizards are susceptible and transmission competent for B. lusitaniae. On the other hand, a lack or low prevalence of other genospecies in ticks that had fed on lizards may suggest that these genospecies could be negatively selected against by green lizards. A similar suppressive sup·pres·sive  
adj.
Tending or serving to suppress.

Adj. 1. suppressive - tending to suppress; "the government used suppressive measures to control the protest"
 effect of Madeiran wall lizard (Podarcis dugesii) on the transmission of spirochetes was observed (33). Borreliacidal activity against B. burgdorferi s.s. was observed in the lizards S. occidentalis and E. multicarinata in North America (15,16). These findings add to the growing support for the hypothesis that there are Borrelia species-specific associations with specific reservoir host species that result from Borrelia species-specific interactions with host serum complement (29).

Significant differences were found in B. lusitaniae prevalence in fed larvae compared with questing nymphs (p [less than or equal to] 0.001, df = 1); none of 183 examined nymphs was infected by this genospecies. This finding raises the questions of whether borreliae are eliminated during molting molting, periodical shedding and renewal of the outer skin, exoskeleton, fur, or feathers of an animal. In most animals the process is triggered by secretions of the thyroid and pituitary glands.  and thus do not contribute to the transmission cycle or whether we were just unable to detect it. Significant differences were found in B. lusitaniae prevalence also in fed nymphs compared with questing adults (p [less than or equal to] 0.01, df = 1). The infection prevalence decreased from 74.5% in fed nymphs to 5% in questing adults. Reduction of infection prevalence has been observed in B. afzelii from 47% in nymphs engorged en·gorge  
v. en·gorged, en·gorg·ing, en·gorg·es

v.tr.
1. To devour greedily.

2. To gorge; glut.

3. To fill to excess, as with blood or other fluid.

v.intr.
 on the rodents to 7% in questing nymphs (9).

The occurrence of B. lusitaniae in ticks is frequent in some areas of the Iberian Peninsula and North Africa, where the organism often represents the only species of B. burgdorferi s.l. complex (13,34). In the rest of the Europe, it has been isolated or detected less frequently, with low prevalence in ticks (30,35,36). The prevalence of B. lusitaniae is the highest in southern Europe and can be exported to other areas by hosts such as birds (37). The 5S-23S rDNA and flagellin sequences of B. lusitaniae-positive ticks and skin biopsy specimens in our study were 100% identical to the B. lusitaniae strain Tr213 from a tick in Turkey (38). The distribution of this borrelial species may be associated with the distribution range of reservoir hosts, including lizards, that inhabit drier and warmer areas. These ecosystems are less abundant in central Europe than in the Mediterranean. Thus, lizards may influence the transmission cycle of borreliae in some localities in which they are the predominant host for ticks. In our study, we found B. lusitaniae in skin biopsy specimens and ticks that fed on green lizards. These findings implicate im·pli·cate  
tr.v. im·pli·cat·ed, im·pli·cat·ing, im·pli·cates
1. To involve or connect intimately or incriminatingly: evidence that implicates others in the plot.

2.
 this species of lizard in the transmission cycle of B. lusitaniae. The competence of other lizard species that feed ticks should be also investigated. The low prevalence of B. lusitaniae in questing ticks, however, indicates that the ecology of B. lusitaniae in endemic foci of central Europe is more complex. Further studies that analyze the circulation of B. burgdorferi s.l. among a broader spectrum of host species should be undertaken.

Acknowledgments

We thank Jean Tsao and Slavka Barlakova for their critical reading of the manuscript and helpful comments and Marcela Mirekova and Renata Ivanova for technical assistance.

This work was partly supported by the Slovak Agency of Research and Development, APVV-51-009205 project (M.D.), VEGA 2/6163/26 to (B.P.), and VEGA 1/1284/04 (I.M.).

Mrs Majlathova is a doctoral student at the Parasitological parasitological

pertaining to or emanating from parasitology.


parasitological examination
includes examination of feces for protozoa, worm eggs or larvae and for tapeworm segments, skin scrapings for arthropod parasites, blood
 Institute of the Slovak Academy of Sciences The Slovak Academy of Sciences SAV (in Slovak Slovenská akadémia vied) is the main scientific and research institution in Slovakia fostering basic and strategic basic research. It was founded in 1942, closed after WWII, and then refounded in 1953. . Her research interests lie in ecology and epidemiology of tickborne pathogens.

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To place in or come into mutual relationship.



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fence lizard - spiny lizard often seen basking on fences in the United States and northern Mexico
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One of the spatial arrangements of atoms in a molecule that can come about through free rotation of the atoms about a single chemical bond.
 polymorphism polymorphism, of minerals, property of crystallizing in two or more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are brookite, anatase (or octahedrite), and rutile.  analysis of the rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from the Czech Republic. Appl Environ Microbiol. 2003;69:509-16.

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The cellular molecules DNA and RNA that act as coded instructions for the production of proteins and are copied for transmission of inherited traits.
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Viktoria Majlathova, * Igor Majlath, ([dagger]) Marketa Derdakova,

* ([double dagger]) Bronislava Vichova, * and Branislav Pet'ko *

* Parasitological Institute of Slovak Academy of Sciences, Kosice, Slovakia; ([dagger]) University of P.J. Safarik in Kogice, Kosice, Slovakia; and ([double dagger]) Institute of Zoology The Institute of Zoology (IoZ) is the research division of the Zoological Society of London (ZSL). It is a government-funded research institute specialising in scientific issues relevant to the conservation of animal species and their habitats.  of Slovak Academy of Sciences, Kosice, Slovakia

Address for correspondence: Viktoria Majlathova, Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Kosice, Slovakia; email: majlat@saske.sk
Table 1. Variability of Borrelia burgdorferi sensu lato
in ticks collected on lizards

                           No
            No. ticks   positive
Stage       examined    ticks (%)

Larvae      197         30 (15.2)
Nymphs      267         47 (17.6)
Total       464         77 (16.6)

              No. ticks positive for genospecies
                     (% positive ticks)

Stage       B. lusitaniae   B. afzelii   B. garinii

Larvae        26 (86.7)      1 (3.3)       1 (3.3)
Nymphs        34 (74.5)      5 (10.6)      1 (2.1)
Total         60 (77.9)      6 (7.8)       2 (2.6)

                       No. ticks positive for genospecies
                              (% positive ticks)

                                                    B. lusitaniae +
Stage       B. burgdorferi s.s.   B. valaisiana   B. burgdorferi s.s.

Larvae             1 (3.3)            1 (3.3)             0
Nymphs             2 (4.3)               0             5 (10.6)
Total              1 (1.3)            1 (1.3)          5 (6.5)

Table 2. Specific infectivity ([I.sub.s]) and host-to-tick
transmission coefficient ([beta].sub.H-T])

                                      Males

Genospecies                 [I.sub.s]   ([beta].sub.H-T])

Borrelia burgdorferi s.l.    0.0477           0.605
B. lusitaniae                0.0377           0.518
B. garinii                   0.0012           0.01
B. afzelii                   0.0012           0.01
B. burgdorferi s.s.          0.0012           0.01

                                     Females

Genospecies                 [I.sub.s]   ([beta].sub.H-T])

Borrelia burgdorferi s.l.    0.0221           0.571
B. lusitaniae                0.0221           0.571
B. garinii                     --              --
B. afzelii                     --              --
B. burgdorferi s.s.            --              --

                                    Total

Genospecies                 [I.sub.s]   ([beta].sub.H-T])

Borrelia burgdorferi s.l.    0.0697          0.5753
B. lusitaniae                0.0597          0.5263
B. garinii                   0.0012           0.01
B. afzelii                   0.0012           0.01
B. burgdorferi s.s.          0.0012           0.01

Table 3. Variability of Borrelia burgdorferi sensu lato
in questing ticks

                      No.
          No. ticks   positive
Stage     examined    ticks (%)

Nymphs      183       31 (19.6)
Females      71       16 (22.5)
Males        71       16 (22.5)
Total       325       63 (19.3)

            No. ticks positive for genospecies
                 (% of positive ticks)

Stage     B. afzelii   B. garinii   B. valaisiana

Nymphs    13 (41.9)    12 (38.7)     2 (6.4)
Females    4 (25)       4 (25)       3 (18.7)
Males      2 (12.5)     5 (31.3)     2 (12.5)
Total     19 (30.2)    21 (33.3)     7 (11.1)

                    No. ticks positive for genospecies
                         (% of positive ticks)

                                                B. valaisiana +
Stage     B. burgdorferi s.s.   B. lusitaniae      B garinii

Nymphs          3 (9.6)              0 (0)           1 (3.2)
Females         3 (18.7)           2 (12.5)           0 (0)
Males           2 (12.5)           5 (31.3)           0 (0)
Total           8 (12.6)           7 (11.1)          1 (1.5)
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Title Annotation:RESEARCH
Author:Pet'ko, Branislav
Publication:Emerging Infectious Diseases
Date:Dec 1, 2006
Words:5550
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