Oddělení fyziologie a imunologie živočichů
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Oddělení fyziologie a imunologie živočichů |
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Náš izolát Borrelia burgdorferi s. l. z klíštěte Ixodes ricinus. Fotografie zhotovil Ústav histologie a embryologie, MU Lékařská fakulta, Brno ČR:
Ixodes ricinus:
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Vedoucí laboratoře: RNDr. Alena Žákovská, Ph.D. Cíle naší práce: Cílem našeho projektu je studium různých aspektů onemocnění Lymeská borrelióza. Tato práce se týká sledování výskytu spirochet se zvláštním zřetelem na patogenní B.b. u Ceratopogonidae, Simuliidae, Culicidae a Ixodidae na několika lokalitách v ČR se zaměřením na bližší identifikaci jejich antigenních struktur, objevení nových Ag struktur, objasnění výskytu těchto patogenních spirochet i v jiných přenašečích než klíšťatech Ixodes ricinus, zjištění eventuální možnosti přenosu přes různé vektory (hlodavci) až na člověka. Chceme též sledovat výskyt antiborreliových Ab u těchto jednotlivých vektorových populací včetně člověka. Dílčí úkoly:
Užité metodiky MIKROSKOPIE V TEMNÉM POLI je metoda na pozorování velmi malých objektů (prvoci, bakterie) a jejich struktur zaživa. Všechny touto metodou pozitivní homogenizáty jsou potom analyzovány metodou PCR. PCR Borrelie v homogenizátech jsou určovány přímo metodou PCR po homogenizaci těl hmyzu, larev a kukel komárů v 0,9% roztoku NaCl.
ODCHYT A VYŠETŘENÍ HLODAVCŮ Na každé lokalitě se odchytávají divoká zvřata (např. terrestriální hlodavci Rodentia) do sklápěcích pastí. Mrtvá zvířata jsou pak pitvána. Srdce každého jedince bude uloženo do 0,9% roztoku NaCl. Získané vypláchnuté srdce bude dále analyzováno ELISA metodou na přítomnost antiborreliových protilátek. Další části orgánů - ledviny a slezina - jsou uloženy do BSK-H media na získání izolátů spirochet. Exoparasité, drobní roztoči a blechy jsou také sbírány z odchycených hlodavců. Jednotlivé vzorky jsou vyšetřeny mikroskopickou technikou v temném poli. METODA ELISA (ENZYM LINKED IMMUNOSORBENT ASSAY) Je metodou užívanou k určení širokého spektra antigenů a protilátek. METODA SDS-PAGGE Všechny isoláty získané ze spirochet jsou testovány na proteinové složení, identifikovány a určeny do druhu srovnáním se standardními kmeny B.b. sensu stricto, B. afzelii, B. garinii, B. lusitanie. METODA WESTERN BLOTT V naší laboratoři bude také zavedena nová metoda, která se používá k identifikaci jakýchkoliv bílkovin, nacházejících se ve směsi. Práce probíhá ve třech laboratořích. Jedná se o laboratoř imunologie, fyziologie a psychofyziologie na katedře Srovnávací fyziologie živočichů a obecné zoologie, která má 3 místnosti. V první místnosti se provádí hlavně metoda PCR, veškeré vybavení (jako např. PCR-cyklu) se vztahuje k této metodice. Další místnost je určena pro speciální imunologické techniky jako je detekce metodou ELISA, analýza pomocí zástinové mikroskopie. V poslední místnosti se pracuje s přístroji nutnými pro SDS-PAGGE elektroforézu. V této laboratoři máme v úmyslu pracovat na těchto úkolech:
Na této problematice se podílí následující pracovníci: Vedoucí výzkumný pracovník RNDr. Alena Žákovská, Ph.D. má více než desetileté zkušenosti s imunologií, obecnou zoologií a také metodami molekulární biologie. Vyučuje a prakticky vede předměty obecná imunologie, obecná zoologie a speciální imunologie. Od roku 1995 se zaměřila na problematiku patologie a epidemiologie Lymeské borreliózy, infekčního onemocnění, které přenáší patogenní mikroorganismus B.b. Kromě vedení projektu se zaměří na metodu ELISA ke stanovení antiborreliových protilátek přítomných v lidských sérech a v sérech divoce žijících zvířat. Prof. RNDr. Jan Knoz, Csc., je hlavní odborník v oblasti obecné zoologie a biologie. Ekologie krev sajících členovců a patogenních mokroorganismů je hlavním zaměřením prací RNDr. Jiřího Halouzky, Csc., který je vedoucím Oddělení medicínské zoologie na Ústavu biologie a obratlovců AVČR ve Valticích. Toto oddělení se bude účastnit projektu spoluprací na izolaci a kultivaci mikroorganismů a přípravě antigenů Mgr. Omar Šerý, Ph.D. pracuje jako asistent. Přes 8 let se věnuje metodě PCR. Antigenní protilátky patogenních spirochet B.b. u dvoukřídlého hmyzu a roztočů (Ixodidae) jsou předmětem doktorského studia Lucie Čapkové, Silvie Šikutové a Kateřiny Pejchalové. Hlavní náplní diplomové práce Evy Janouškovcové je pak analýza bílkovinného složení za účelem charakteristiky spirochet v rámci rodu, na dané problematice pracují ve svých diplomových pracích i Karel Vostál, Gabriela Rašková, Petra Novotná, Alena Holíková. Our
research work presented here in the following text is included in „Spatial
and Temporal Biodiversity Dynamics in Ecosystems of Central Europe“ research
programme MSM
143100010 (formerly CEZ: J07/98:143100010) - VZ MU 0429, coordinator: J. Vaňhara
(Department of Zoology and Ecology)
and represents the results of our work from 1999-2004 Study of some aspects of Lyme borreliosis disease from the point of view
of immunology and zoology
Alena Žákovská „Lyme
borreliosis is a fascinating disease, the aetiopathology of which is not yet
completely known. Different subspecies of Borrelia
burgdorferi sensu lato are responsible for the variable clinical course of
the disease. Optimal therapy of LB is still lacking, but doxycycline,
amoxicillin, penicilin and ceftriaxone are recommended most frequently (Lippincot
&Wilkins)”. Introduction Lyme borreliosis is a multisystem infection caused by the spirochaete Borrelia burgdorferi which is transmitted by the usually asymptomatic bite of certain ticks of the genus Ixodes (Lane et al., 1991). The history of this disease started in Europe at the end of the last century and continues up to the present day. In the beginning of the 1980s Lyme borreliosis seemed to be relatively rare disease, but within a few years it became clear that it was a false assumption. Because of this high incidence, clinical presentation that differs in North America and Eurasia, diagnostic difficulties, problem with evaluating the results of treatment and other complication, this disease is one of the most discussed infection (Hercogova &Brzonova). Epidemiology Lyme disease is caused by the tick-borne spirochaete B. burgdorferi sensu lato which has been subdivided into eleven genospecies, only three of them are regarded to be responsible for the Lyme disease: B. burgdorferi sensu stricto, B. afzelii, B. garinii (Gern&Falco, 2000). In the USA only the first species is involved, whereas strains of all three species have been isolated from patients in Europe (Rowe, 2000). Ticks of the genus Ixodes are the vectors that transit the infection to mammals in endemic areas: the North America and Eurasian continents (O`Connell et al. 1998, Steere 1994, Sood, 1999). LB was recorded from more than 50 countries in the world and mostly appeared in the North hemisphere. In Europe more than 50.000 cases is anually estimated. Austria, Slovenia, Sweden and the Czech Republic belong to the most highly endemic areas (Hercogova&Brzonova). Czech Republic with its geographical character is very suitable biotope for the presence of the most frequent vector Ixodes ricinus (L.), and belongs with in average 3.500 cases of LB disease to the most struck countries. The patient’s evidence is involved from 1985 under the diagnosis A 69.2. The highest number of LB disease cases was recorded in 1995 (6302 cases). Incidence of the tick-borne diseases depends on climatic variations, and the milder climate of the 1990s (compared to 1980s) has made increases in their incidence and the northward expansion of the geographic distribution possible (Hercogova &Brzonova, Kraigher et al. 1997). Lyme borreliosis disease LB
is a multisystem disorder, which may affect mainly the skin, the neurological
system and the joints (Bennet, 1995). Three cutaneous manifestations are the
most characteristic and the most specific of the disease and accompany the
three disease stadium which can be developed in untreated case. After the tick
bite the first is a cutaneous lesion “the erythrema migrans”. Slight fever,
myalgia, arthralgia, headache may accompany the erythrema, which disappears
spontaneously after antibiotic treatment. Lymphocytoma is another cutaneous
lesion, located at the ear lobe or nose wing. The third and late manifestation
of acrodermatitis may occur several years after the primary lesion. The
disseminated infection occurs some weeks or months after tick bite. They
involve mainly joints coursing arthritis and neurological system causing
meningitis, radiculo-neuritis and facial palsy (Postic&Baranton, 2001). Erythrema
migrans
http://www.cdc.gov/ncidod/dvbid/lyme/diagnosis.htm Osp
A, OspB, OspC, OspD, OspE and OpsF are the most important genes and have been
well documented as antigens for diagnosis. Some of them vary in size according
to the species. For example OspA found in plasmid being 50kb in Bbss, 55kb in B.
garinii, 56kb in B. afzelii (Bennett,
1995). Some more important
antigens in Bbsl are responsible for variety of characteristics such as P39,
37, HSP 66, 75, DbpA, DbpB, Bdr proteins and so on. Our
research work presented here is included in „Spatial and Temporal
Biodiversity Dynamics in Ecosystems of Central Europe“ research programme MSM 143100010 (formerly CEZ: J07/98:143100010) - VZ MU 0429, coordinator: J. Vaňhara (Department of Zoology and Ecology). Long
– time aim of this work is a study of some aspects of Lyme borreliosis
disease from the point of view of immunology and zoology. The
study presents an interaction between haematophagous arthropods and mammals
reciprocally, depending on the transmission of possible pathogens. Our
research activity was focused on three main branches: 1.
Study of LB vectors (ticks), potential vectors (other haematophagous
arthropods such as mosquitoes, small mites) ecology and epidemiologic
consequences of their pathogens 2.
Study of LB hosts 3. Study of antigenic characteristics of isolated strains of pathogenic borreliae as an etiologic agents of LB. Ad
1 Ticks Ticks play an important role in human and veterinary
medicine, in particular due to their ability to transit a wide spectrum of
pathogenic micro-organism of protozoa, rickettsial, bacterial and viral origin.
New zoonoses are also emerging, some of which are caused by tick-borne
pathogens and these have a serious impact not only on human health, but also
they are responsible worldwide for great economic losses in terms of mortality
of livestock animals (Sparagano et al. 1999). Tick-borne pathogens can co-exist
in the same tick vector or be carried by different tick species, as well as by
hosts and it is difficult to identify a pathogen in carrier animals or ticks
which are carrying low level of infection. In this situation, molecular tools,
particularly amplification of specific markers using the polymerase chain
reaction (PCR) have revolutionized detection and identification of pathogenic
organism (Riipkema et al. 1995; Sparagano et al. 1999). The main vector of
this infectious agent in the Czech Republic is Ixodes
ricinus (L.), which is the most common tick in Europe. Ixodes persulcatus Schultze is another vector in Eurasia. Ixodes
pacificus Cooley and Kohls and Ixodes
scapularis Say are vectors in North America (Anderson, 1989). In
the present survey, Czech Ixodes ricinus
ticks were tested for the presence of Borrelia
spirochaetes and how are the prevalence of ticks dependent on various
conditions. We focused on the distribution of B.
burgdorferi s. l. in southern Moravia (Brno city) and eastern Bohemia
(Vysoké Mýto surrounding). Results from eastern Bohemia were published in
Our interest was also intended in the occurrence of various borrelian genospecies in the tick population, as described in
The infected ticks frequently
occur in peripheral forest parks in the neighbourhood of large cities in
central Europe. Therefore, we were interested mainly in the occurrence and
infestation of ticks by Lyme disease in the urban park surrounding the second
largest city of the Czech Republic - Brno, during 1999 – 2004, because it is
possible for the urban population in Brno to come into contact with the
causative agent of Lyme disease in this park.
In these articles we studied Tick
activity and positivity and
from from these results more articles were finished, such as
In the following articles the PCR as an used modern molecular techniques
appeared
The last survey containing sequenation techniques was described in
Mosquitoes
Culex
(C.) pipiens s.l. http://www.gardensafari.net/first/mosquitoes.htm Some authors such as Hard,
1996; Doby at al. 1986 and Luger, 1990 describe clinical cases of patients
reporting an insect bites after that typicaly erythrema migrans and
antiborrelian antibodies appeared. The diagnosis of Lyme disease follows was
made. These facts open the discussion if biting insects such as deer flies,
black flies and mosquitoes might play a role in the ecology and epidemiology
of Lyme borreliosis (Doby et al., Magnarelli et al. 1986). Spirochetes in blood sucking arthropods have been observed
since the beginning of the 20. century (Jaffé, 1907; Sinton and Shute, 1939;
Masseguin and Palinacci, 1954). In the Czech Republic the evidence of the
presence of spirochetes (including borreliae) has been observed and reported
in works of Jírovec, 1943; Halouzka, 1993; Halouzka et al. 1998; 1999; Hubálek
et al. 1998. The first isolated strains of pathogenic Borrelia from culicine
mosquitoes Aedes vexans and Culex
pipiens molestus were reported from South Moravia, Czech Republic
(Halouzka et al. 1998; 1999). From these fact we wanted to know more about the
circulation of Bbsl (if found) in other haematophagous arthropods than Ixodid
ticks and this was one of our further research aim. Study of the presentation
of spirochaetes and their identification, focused on the pathogenic Bbsl,
found in different growth stadium of mosquito families Culicidae
was presented in the following
articles: For this part of research the using of our developed special molecular
methods was necessary.
Small
mites
Laelaps
agilis Department
of Komparative Snímal Physiology and general zoology This study is intented on a
presence of borreliae in mites (except the ticks) parasiting at small rodents.
Such type of research has not been carried out for the time being and this way
the study tries to complete the knowledge about the possible occurrence of
this spirochaete in other arthropods than ticks in the nature. Ixodid ticks (Ixodida)
and gamasid mites (Mesostigmata) are
the most abundant arthropod groups in ectoparasite communities of rodents in
Central Europe. Both parasite groups are important vectors of various diseases
and infested a very wide host range. The presence of borrelian DNA in other
groups of arthropods than ticks is very rare by described. In this study we
focused our attention on the possible occurrence of borreliae in other
ectoparasites of order Acarina than
ticks. Mites were chosen purposely as the study material because of their
appropriation for the same taxonomic group as ticks – the most frequent
borreliae vectors. We are based on the report, that the presence of some other
pathogenic agents – e.g. Francisella
tularensis causing tularemia or rickettsiae was confirmed at great numbers
of gamasid mites including Haemogamasus
nidi as well (Zuevskii, 1979; Lysý et al. 1999; Kocianová, 1982) and we
try to find other pathogenic agent. The
first very interesting so far nowhere published such a results were described
in
From these data we should be
aware of the fact that this group of ectoparasites may participate in a
transmission of various diseases and from this reason it should be paid more
attention to this problems for the future. Ad
2 Apodemus
sylvaticus http://www.consult-eco.ndirect.co.uk/lrc/specnews.htm As written before, Lyme disease is associated with blood-sucking
arthropods, especially ticks, from which species of genus Ixodes are the most important. European species, transferring Bbsl
are the following: Ixodes ricinus, feeding on at least 317 animal species, Ixodes
hexagonus, parasiting in carnivores and hedgehogs and Ixodes
uriae, involved in transmission of borreliae in seabird colonies (Gern,
2001). About 35 vertebrate species were identified as reservoir hosts of Bbsl.
(it means animals participating in circulation of Bbsl. in nature). Among them
small mammals and birds are the best known host species. Additional vertebrate
species like hedgehogs and rabbits function as reservoir hosts (Gern, 2001).
Deer, domestic animals (cats, dogs) and reptiles (lizards) can also serve as
reservoirs (Dlouhý, 1996). Rodent species, for example Apodemus mice and Clethrionomys
voles, have been studied as typical reservoir hosts of Bbsl in various
enzootic areas in Europe (Matuschka et al. 1992, Kurtenbach et al. 1995).
Microtus
agrestis http://home2.planetinternet.be/rv047190/hoe/muisje.htm Research
work submitted in the report concerned the spirochaete presence and their
identification focused on Bbsl in small rodents of the families Microtidae
a Muridae in some
chosenlocalities in the Czech Republic. The work also includes the study of
immune response to the borrelian infection in these small mammals. This study tries to find the species of reservoir hosts for B.b.s.l.
living in the Czech Republic. What is the rate of obtained immunity against
borrelia and which of borrelian species have infected captured rodents in the
local fauna of these mammals in the Czech Republic
were another questions for our research. The partially answers to the
questions we tried to summarized in the following articles:
Ad3 Borrelia burgdorferi
www.microbelibrary.org/images/jnelson/Images/Borrelia.jpg.2003 Since its discovery in 1982, many strains of Borrelia burgdorferi have been isolated from ticks, humans and
reservoir hosts. To date, B. burgdorferi
can be divided into at least 11 species (B.
burgdorferi sensu stricto, present in Europe and in the USA but rare in
Russia and apparently absent from Asia; B.
garinii, B. afzelii, B. valaisiana
and B. lusitaniae in Eurasia, B.
japonica, B. tanukii and B. turdi
restricted to Japan, B. sinica in
China, and B. andersonii and B.
bissettii in the USA) (Le Fleche et al. 1997, Masuzawa et al. 2001, Postic
et al. 1994, Postic et al. 1998). In addition, several as yet unnamed genomic
groups contribute to the increasingly recognized diversity of B.
burgdorferi sensu lato. Of the 11 different species, only B. burgdorferi sensu stricto, B.
garinii and B. afzelii (Canica
et al. 1993, Péter et al. 1992) are undoubtedly involved in clinical cases of
Lyme disease. However, other than the three well-known human pathogenic
species (Wang et al. 1999), the genospecies A14S may also cause Lyme disease
and B. valaisiana or B. bissettii could be associated with EM. The European isolates are
more heterogeneous with respect to their antigenic profiles than American ones
(Wilske et al. 1993, Wilske et al. 1988), and the most frequent genomic groups
in Europe are B. garinii and B.
afzelii (Hubálek &Halouzka, 1997) Antigens The structure of B.
burgdorferi sensu lato is typical for spirochaete: a spiral or coil-shaped
cell that is generally 20-30μm in width. Individual spirochaetes, however
can vary in length, diameter, tightness and regularity of the coils. The
protoplasmic cylinder containing the cytoplasm with its organelles and
flagellar apparatus is covered by a periplasm and a lipoprotein-based outer
surface membrane. The genome size is relatively small, approximately 1,5
megabases, consisting of a linear chromosome of 950kb and at least 21
extrachromosomal DNA elements or plasmids (620kb within nine linear and 12
circular). Plasmid genes are of specific interest, as they contain many of the
genes associated with spirochaete pathogenicity.
While chromosome genes coding for a basic set of known eubacterial proteins
that drive cell cycle, growth and metabolism, with an unusual absence of genes
responsible for cellular biosynthetic reactions, plasmids encodes 90% genes
have no convicting similarity to genes outside Borrelia genus, suggesting that
they perform specialized functions possibly related to spirochaete adaptation.
Further experiments demonstrated that some of the plasmids can be lost during
propagation of the bacteria in vitro, and loss of infectivity in mice often
parallels the loss of specific plasmids (Pal&Fikrig, 2003, Hefty et al.,
2001). Research work concludes the
following aims: confirmation of the infection agents, which is specifically
concentrated on the identification of isolated strains, identification of antigenic structures using the molecular
biologic methods such as PCR, PCR-RFLP, sequenation, gradient PAGE
electrophoresis, which includes study of spirochaete DNA and proteins,
discovery of a new antigenic structures and reaction of the mammals organisms
on the the presence of variety of antigens. Determination
of spirochete isolates and study of their protein composition until 2000 was
reported in
Identification
of isolated strains with the characterisation of Flagellin gene and protein
composition was the aim of the following articles
Study of immune response to B. afzelii antigen is
involved in the artical
The
last survey containing sequenation technics focused on the flagellin gene of
individual isolated strains was described in
Materials and MethodsThe
work was proved by not only current biology techniques such as collection,
cultivation, dark field microscopy, but also with the methods of modern
molecular biology, as PCR, PCR-RFLP, gradient PAGE, sequenation. In research
work presented here the following methods were used: Tick, mosquitoe,
small mites collection and preparation
The
ticks, all belonging to the species Ixodes
ricinus (L., 1758), were collected in three areas of two different regions
during the years 1999 - 2004. One selected locality is situated in southern
Moravia (Brno-Pisárky, land-register
Brno) and the second locality belongs to a small area of eastern Bohemia (Vysoké
Mýto, l.-r.). The urban park Brno-Pisárky distant 2 km from the city
centre is a part of Brno city. It is situated at 197 - 210 m altitude and
forms a bottom of the Pisárky valley. The second habitat is situated around a
pond, near the town Vysoké Mýto in the western part of the district Ústí
nad Orlicí (East Bohemia),. For all areas deciduous wooden forests are
typical. Ixodes
ricinus
ticks were collected by the flagging method (dragging white flannel flags over
low vegetation). All collected ticks were placed into tubes and stored at 5°C
until examination for the presence of spirochetes. For the examination the
ticks were placed on a watch glass. Mosquitoe
colection Female
mosquitoes of genus Culex, Aedes, Anopheles,
Theobaldia, Culiseta collected
in localities in the surrounding of the town Vysoké
Mýto, Břeclav, l.-r. (South Moravia) and Poodří:
Bažantula (l.-r. Studénka) and Blücherův les (l.-r. Polanka nad Odrou),
(North Moravia) were examined for the presence of spirochetes during the years
1999-2001. In Vysoké Mýto and Břeclav “overwintering” mosquitoes were
collected by an aspirator in their typical shelters, i. e. cellars of several
village buildings, from November to March. Summer mosquitoes in Vysoké Mýto
and Studénka were collected using a clap – net. The samples were kept alive
under cool (5°C) and humid (r. h. 90 %) conditions until processing.
Mosquitoes were dissected and their abdomen content triturated individually on
a slide in a drop of saline solution. During
1999-2001, larvae of Culex (C.) pipiens s.l. of all four instars were
collected from a pond at Brno-Žebětín, Brno-Obřany (South Moravia), Vysoké
Mýto (East Bohemia) by using a beaker. The locality Brno–Žebětín
is situated in the west part of Brno city (in the edge of forester district
Podkomory). It is a part of Brněnská kotlina basin. The larvae were
collected from the rest of pond and wet ground near confluence of “Žebětínský”
and “Vrbovecký” stream. Brno-Obřany
locality is situated northwest part 2km off Brno city about 350 m above sea
level, in the hill slope. This area is used as a little garden among other
ones with fruit trees. All larvae were collected from the barrel with
rainwater. Larval abdominal content was examined and triturated on slides in a
drop of phosphate-buffered saline and and prepared for dark field microscopy (DFM).
Small
mite preparation Entrapped
rodents from the locality Poodří:
Bažantula (l.-r. Studénka) and Blücherův les (l.-r. Polanka nad Odrou)
were
examined by a pincette and parasitic mites of order Acarina
were collected from its hair. Every mite was dissected using a microscope and
a preparation needle. At dissecting process it was very important not to
damage the parasite too much, because some morphological signs are neccessary
for the right determination and their absence would make the identification of
the sample impossible. After removing of the guts the dissecting mites were
kept in 70% ethanol until the later determination that was carried out only in
cases positive for the presence of DNA of B. burgdorferi s.l. To be
identified each pattern was closed into a drop of a medium containing the
chloralhydrate on a slide a recovered with a sterile glass. At the
determination of mites the solutions with a content of this matter are used
because of its good shine – through character (Novák &Povolný, 1969),
in our case the Hoyer´s was used. Dark
field microscopy Midgut
tissues from each tick, small mites and mosquitoe and larvae abdomen content
were removed by dissection, suspended in a drop of saline, and examined by
dark-field microscopy at 200x magnification.
All
dark-field positive samples were PCR tested independently in two laboratories
by the following method: Single-tube nested PCR Isolation
attempts Spirochetes
in positive DFM samples were counted, and the highly infected tick tissues
containing more than 100 spirochetes were transferred into BSK-H medium
(Sigma) supplemented with 5% rabbit serum (Sigma) and antibiotics (e.g.
rifampicin 50 mg/ml,
phosphomycin 100 mg/ml)
to prevent bacterial contamination for isolation attempts. Cultures were
incubated at 33°C
for six weeks and examined for the presence of spirochetes by dark-field
microscopy once per week. When no spirochetes were detectable in the test
culture or spirochetes did not multiply, the culture was eliminated.
Spirochete cultures were harvested by centrifugation (10 000 x g, 15 min),
washed three times in phosphate-buffered saline, resuspended in saline and
stored frozen until being used for identification of Borrelia
strains by PCR-RFLP detection and SDS-gradient PAGE characterization. Rodent
trapping Trapping
was practised in 7
localities. In two localities in Poodří
characterized by floodplain forests (Querco-Ulmetum alnetosum association,
Fraxinus excelsior, Alnus glutinosa), in Rokytná
(l.-r. Vémyslice and Tulešice) on wooded bank of Rokytná river (Robinia
pseudoacacia, Populus tremula, Acer campestre, Sambucus
nigra) and in Valchov (l.-r.
Valchov) near mixed wood (Populus tremula, Carpinus betulus, Larix
deciduas, Picea abies), in Bílé
Karpaty (l.-r. Malá Vrbka) in the part of wood (Tilia
cordata, Carpinus
betulus, Sambucus nigra,
Swida sanguinea)
and meadow, in Dlúhý Hrúd (l.-r.
Lanžhot) in floodplain near confluence of Dyje and Morava rivers (Carpinus betulus, Quercus
rubra, Acer campestre,
Sambucus nigra),
in Jansův vršek (land-register
Kamenice u Jihlavy) on mixed wooded slope (Picea
abies, Pinus silvestris, Carpinus
betulus, Acer campestre). Rodents
were obtained by trapping with snapping spring traps. In some cases rodents
were trapped with „life-hunt“ traps. The snapping traps were baited with a
piece of wick fried with lard and „life-hunt” traps were baited with a
piece of fish covered by cotton wadding. Traps were placed on solid ground in
line at a distance of 7 m from each other. They were opened at least 2 days
and checked every 12 hours. Trapped rodents were collected and transferred to
the laboratory for next investigation early in the morning. Autopsy was
carried out during followed 3 hours from morning to noon. The rodents were
identified and sexed. Heart
rinses and sera, as samples were prepared from rodents. The heart of dead
individual was put into 0,85% physiological solution for a period of 1-2 days
at temperature of 4°C. After removing the heart the remaining solution was
centrifuged and drained supernatant stored at -18°C. Blood taken from neck
artery of anesthetized living mouse was stored at 4°C overnight. Next day the
serum was separated and stored at -18°C. ELISA From
the year of 2001 -2004 home-made ELISA with antimouse IgG was practised. The
whole cell culture from B. afzelii (BRZ
16 - strain isolated from I. ricinus
from the Pisárky park area in Brno, southern Moravia) was used for coating
Sera and heart-rinses were examined by a modified ELISA, available in
commercial sets (TestLine, Prague) used for diagnosis of Lyme borreliosis in
human medicine, in the following way: Microplates were parallelly filled with
100 ml of respective antigen diluted in carbonate buffer at pH 9.6 (2 mg/ml)
and incubated overnight at 4°C. After washing three times with phosphate
buffer (pH 7,4) containing 0,05 % Tween 20, 100 ml portions of sera diluted at
1:100 in phosphate buffer with 0.05 % Tween 20 and 0.3 % cassein and the same
portions of heart-rinses (undiluted) were added and incubated at 37°C for 1
hour. After triple washing of the plates, 100 ml portions of anti-mouse IgG
peroxidase conjugate (Sigma) were added per well, diluted at 1:2000. After 1
hour of incubation and a subsequent washing, 100 ml per well of substrate
solution (0.1 M citrate buffer pH 4.7-5.0 with 0,05% H2O2)
with orthophenylene diamine were added. The reaction was stopped with 1 M H2SO4
after 10 min of incubation. The absorbance was measured at 492 nm. The serum
from a wild immunized mouse was used as a positive control. As negative
control were used the wild mouse sera negative to antigens (OD492 <
0.300). Samples with OD492 > 0,300 were considered positive.
Elisa
results PCR-RFLP
detection DNA
purification.
DNA of detected samples was isolated from homogenates using a DNA isolation
kit (Malamité v.o.s., Czech Republic). This procedure is based on cell lyses
by sarkosyl and chaotropic ions and subsequent binding of DNA to silica
particles. DNA was eluted from silica particles in 20 ml
of TE buffer (10mM Tris-Cl, 1mM EDTA). Volume of 5 ml
of this preparation was used for amplification. PCR
assay
based on the specific flagellin sequence amplification for detection of B. burgdorferi sensu lato was performed (Picken et al. 1996). The 50
ml
PCR mixture contained: 1x HotStarTaq Master Mix (Qiagen, Germany), 15 pmol of
each FL3 primer (5’-mga gct tct gat gat gct gct ggy atg ggr g-3’) and FL5 primer
(5’-grg gaa ctt gat tag cyt gyg caa
tca ttg cc-3’), 100 mM
of dUTP (Sigma), and 5 ml
of template DNA received after standard DNA isolation. All PCR runs were
performed on a thermocycler (PTC-200, MJ Research) with the following profile:
an initial activation step at 96°C for 12 min, thirty cycles consisting of a
denaturation step for 10 sec at 96°C, an annealing step for 10 sec at 65°C,
an extension step for 40 sec at 72°C and the final extension at 72°C for 4
min.
PCR
analysis results Restriction
fragment length polymorphism (RFLP). The
sequences coding flagellin gene (fla)
of Borrelia burgdorferi sensu
stricto, Borrelia afzelii and Borrelia
garinii were gathered from GeneBank database (NCBI). The multiple
alignments were by the CLUSTALW program (Thomson, Higgens and Gibson 1994)
done and the restriction fragment length polymorphism (RFLP) patterns,
obtained after AluI endonuclease
digestion, were predicted using the WEBCUTTER 2.0 program. These predicted
RFLP patterns were proved on reference Borrelia
burgdorferi sensu lato strains achieved from German Collection of
Microorganism and Cell Cultures (DSMZ). For identification of Borrelia
strains only fragments longer than 100 bp were taken into account. Restriction
analysis
of amplified PCR products was performed by AluI
endonuclease digestion (New England BioLabs). The restriction DNA
fragments were analysed by agarose gel electrophoresis through 2% agarose gel,
visualized by ethidium bromide staining, detected using UV transillumination
(312 nm), and analysed by ULTRA LUM gel detection and analysis system (Ultra-lum,
Inc.).
PCR-RFLP
analysis results SDS-PAGE
electrophoresis For
our purpose we used a slightly modified SDS-gradient PAGE test of whole
cells Borrelia according to Laemmli [21]. The protein
concentration was estimated by the method of Bradford [5]. Spirochete
samples were diluted in buffer, pH 6.8 (diluted by H2O 1:3), to the
protein concentration of 3 mg/ml. 10 ml
of diluted sample, 10 ml
of sample buffer (0.5M Tris-HCl, 0.02M EDTA, 1.46M saccharose, 3.7% SDS, 0.03%
bromphenol blue), 7 ml
of merkaptoethanol and 73 ml
of buffer pH 6.8 (diluted by H2O 1:3) were mixed. These prepared
samples were incubated (60 min. at 60 °C), centrifugated (4000 x g, 10 min)
and separated by electrophoresis into 7.5% - 20.02% gradient gel in the
surrounding of Tris-glycin-EDTA, pH 8.3 at voltage 400 - 610 V. For the
protein visualization in the gels all gels were stained with AgNO3
by Rabilloud [32] and each of the protein samples was evaluated using
videodensitometr and Molecular Analyst software (Bio-Rad) with Broad Range
comparative standard (Bio-Rad). The high passage strains B. burgdorferi
sensu stricto (B-31), B. afzelii (Br-75) [15] and B. garinii (Br-97
isolated from I. ricinus female tick, locality Valtice, 1995, Czech
Republic, personal message) were used as references.
SDS
gradient PAGE results References Anderson
J. Epizootiology of Borrelia burgdorferi in Ixodes tick vector
and reservoir hosts. Rev Infect Dis
1989;11:1451-1459. Bennett CE. Ticks and Lyme disease. Adv Parasitol 1995;36:343-405. Bradford, MM. Detection of
the protein concentration by a spectrophotometry. Analytical Biochemistry
1976;72:248-254. Canica, MM, Nato, F, du
Merle, L, Mazie, JC, Baranton, G, Postic, D. Monoclonal antibodies for
identification of Borrelia afzelii sp. nov. associated with late
cutaneous manifestations of Lyme Borreliosis. Scand J Infect Dis 1993;
25:441-448. Doby JM, Anderson JF,
Couatarmanac´h A., Magnarelli LA and Martin A.
Lyme disease in Canada with possible transmission by an insect. Zbl.
Bakt 1986;A 263: 488–490. Dlouhý
P. Lymeská borrelióza v praxi. Psychiatrické
centrum, Praha, 1996. Gern
L. Borrelia burgdorferi sensu lato in Europe and North America: two
different ecological situations. In: International conference on Lyme
borreliosis, Bruxelles:
Rily group, Ottignies, Belgium, 1-5, 2001. Gern
L. & Falco RC. Lyme disease. Rev
sci tech Off int Epiz. 2000;19:121-135 Halouzka
J. Borreliae in Aedes vexans and hibernating Culex pipiens molestus
mosquitoes. Biologia 1993;48/2:123–124. Halouzka J, Postic D, Hubálek
Z. Isolation of the spirochaete Borrelia afzelii from the mosquito Aedes
vexans in the Czech republic. Med Vet Entomol 1998; 12:103–105. Halouzka J,
Wilske B, Stünzner D, Sanogo YO, Hubálek Z. Isolation of Borrelia afzelii
from overwintering Culex pipiens biotype molestus mosquitoes. Infection
1999; 27: 4-5, 275–277. Hard S. Erythema
chronicum migrans (Afzelii) associated with mosquito bite. Acta Derm Venerol
1966; 46: 473–476. Hefty
PS, Jolliff SE, Caimano MJ, Wikel SK, Radolf JD, Akins DR. Regulation of Osp-E-Related,
OspF-Related and Elp Lipoproteins of Borrelia burgdorferi Strain 297 by
Mammalian Host-Specific Signals. Infect. Immune. 2001;69:3618-3627. Hercogova J&Brzonova
I. Lyme disease in central Europe. Curr Opin Infect Dis 2001;14:133-137. Hubálek Z, Halouzka J.
Distribution of Borrelia burgdorferi sensu lato genomic groups in
Europe, a review. Eur J Epidemiol 1997; 13:951-957. Jaffé J.
Arch. Protistenkd 1907;9: 100–107. Jírovec O.
Cizopasní prvoci v larvách našich muchniček. Příroda
1943; 26: 289-294. Kocianová E. Nest
ectoparasites (gamasid mites) as vectors for rickettsia under experimental
conditions. Parazitologia 1982; 16(3):
219 – 3. Kraigher A, Hočevar GA,
Klavs I, Marinič FN, Pahor L, et al (1997) in: Zore A, Petrovec M, Prosenc K,
Trilar T, Ružić-Sabljić E, Avšič-Županc T. Infection of small mammals
with Borrelia burgdorferi sensu lato in Slovenia as determined by
polymerace Chin reaction (PCR). Sien Klin Wochenschr 1999;111/22-23:997-999. Kurtenbach
K, Kampen H, Dizij A, Arndt S, Seitz HM, Schaible UE, Simon MM. Infestation
of rodents with larval Ixodes ricinus (Acari: Ixodidae) is an
important factor in the transmission cycle of Borrelia burgdorferi s.l.
in German woodlands. J Med Entomol 1995; 32(6): 807-817. Laemmli, UK. Cleavage of
structural proteins during the assembly of the head of bacteriophage T. Nature
1970;227:680‑685. Lane
RS, Piesman J, Burgdorfer W. Lyme
borreliosis: Relation of its causative agent to its vector and hosts in North
America and Europe. Annual Review of Entomology 1991;36: 587-609. Le Fleche A, Postic D,
Girardet K, Peter O, Baranton G. Characterisation of Borrelia lusitaniae
sp. nov. by 16S ribosomal DNA sequence analysis. Int J Syst Bacteriol 1997;
47:921-925. Lippincot
W &Wilkins. Curr Opin Infect Dis 2001;14:133/137.
Luger SW.
Lyme disease transmitted by a biting fly.
New Eng J Med 1990;Vol.
322, 24:1752. Lysý J, Nosek J,
Vyrosteková V, Kovacik J. Isolation of Francisella tularensis from mites Haemogamasus nidi
and Laelaps hilaris in western Slovakia. Med Parazitol (Mosk.) 1999; Apr – Jun (2) : 26 – 30. Magnarelli LA,Anderson JF,
Barbour AG. The Etiologic Agent
of Lyme Disease in Deer Flies, Horse Flies, and Mosquitoes. J Infect Dis Vol
1986; 154, 2 :355–358. Masseguin A&Palinacci
A. Bull Soc Pathol Exot 1954; 47: 3. Masuzawa T, Takada,N,
Kudeken M, Fukui T, Yano,Y, Ishiguro F, Kawamura Y, Imai Y, Ezaki T. Borrelia
sinica sp. nov., a Lyme disease-related Borrelia species isolated in
China. Int J Syst Evol Microbiol 2001; 51:1817-1824. Matuschka
FR, Fischer P, Heiler M, Richter D, Spielman A. Capacity of European
animals as reservoir hosts for the Lyme disease spirochete. Journal of
Infectious Diseases 1992; 165: 479-483. Novák K& Povolný D.
Metody sběru a preparace hmyzu. Academia Praha1969;99 – 100. O`Connell
S, Granstrom M, Gray JS, Stanek G. Epidemiology of European Lyme Borreliosis.
Zbl Bakt 1998; 287:
229-240. Pal U &
Fikrig E. Adaptation of Borrelia burgdorferi in the vector and
vertebrate host. Microbes and Infection 2003; 5: 659-666. Péter O& Bretz A. Polymorphism
of outer surface proteins of Borrelia burgdorferi as a tool for
classification. Zbl Bakt 1992; 277:28-33. Picken MM, Picken RN, Han
D, Cheng, Y, Strle F. Single-Tube Nested Polymerase Chain Reaction Assay Based
on Flagellin Gene Sequences for Detection of Borrelia burgdorferi sensu
lato. Eur J Clin Microbiol Infect Dis 1996; 15:489-498. Postic D, Assous MV,
Grimont PAD, Baranton G. Diversity of Borrelia burgdorferi sensu lato
evidenced by restriction fragment length polymorphism of rrf (5S) rrl (23S)
intergenic spacer amplicons. Int J Syst Bacteriol 1994; 44:743-752. Postic D, Ras NM, Lane RS,
Hendson M, Baranton G. Expanded Diversity among California Borrelia isolates
and description of Borrelia bissettii sp. nov. (formerly Borrelia group
DN127). J Clin Microbiol 1998; 36:3497-3504. Rabilloud T, Carpentier G,
Tarronx P. Improvement and simplification of low-background silver staining of
proteins by using sodium dithionite. Electrophoresis 1988;9(6):288-291. Riipkema SGT, Molkenboer
MJCH, Schouls LM, Jongejan F, Scellekens JFP. Simultaneous detection and
genotyping of three genomic group Of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus
ticks by characterization of the amplified intergenic spacer region between 5S
and 23SrRNA genes 1995. J Clin Microbiol;33: 3091-3095. Rowe PM. Chronic Lyme
Disease: the debate goes on. Lancet 2000;355:1435. Sinton J A. Shute PG.
Spirochaetal infections of mosquitoes. The J Trop Med
Hyg.1939; 42: 9,p.03/05. Sood
SK. Lyme disease. Pediatr Inf Dis 1999;18:913-925. Sparagano
OAE, Allsopp MTE P, Mank RA, Rijpkema SG T, Figureoa JV, Jongejan F. Molecular
detection of pathogen DNA in ticks (Acari: Ixodidae): A Reviw Exp Appl Acarol 1999;23: 929-960. Steere
AC. Lyme disease: a growing threat to urban populations. Proc
Natl Acad Sci USA1994; 91:
2378-2383. Wang G, van Dam AP,
Dankert J. Phenotypic and genetic characterization of a novel Borrelia
burgdorferi sensu lato isolate from a patient with Lyme borreliosis. J
Clin Microbiol 1999; 37:3025-3028. Wilske B, Preac-Mursic V,
Göbel UB, Graf B, Jauris-Heipke S, Soutschek E, Schwab E, Zumstein G. An OspA
serotyping system for Borrelia burgdorferi based on reactivity with monoclonal
antibodies and OspA sequence analysis. J Clin Microbiol 1993;
31:340-350. Wilske B, Preac-Mursic V,
Schierz G, Kűhbeck R, Barbour AG, Kramer M. Antigenic variability of Borrelia
burgdorferi. Ann N Y Acad Sci 1988; 539:126-143. Zuevskii A .: Role of
gamasids in the epizootiology of tularemia. Zentralbl
Bakteriol (Orig. A) 1979;
Jul 244 (2- 3): 324 – 6.
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