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:
- Study of LB vectors (ticks), potential vectors (other haematophagous arthropods such as mosquitoes, small mites) ecology and epidemiologic consequences of their pathogens
- Study of LB hosts
- Study of antigenic characteristics of isolated strains of pathogenic borreliae as an etiologic agents of LB.
Ad 1
Ticks
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
Čapková L., ®ákovská A., Knoz J., Dendis M., ©erý O., 2002: Further spirochaetes findings in Culex sp., Aedes sp. mosquitoes and in Ixodes ricinus ticks. Biológia.57;3:389-394.
Our interest was also intended in the occurrence of various borrelian genospecies in the tick population, as described in
Janouąkovcová, E., ®ákovská, A., Halouzka, J., Dendis, M., 2004: Occurrence of Borrelia afzelii and Borrelia garinii in Ixodes ricinus Ticks from Southern Moravia, Czech Republic. Vector Borne and Zoonotic Dis.4 (1):43-52.
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
®ákovská A, 2000 a: Monitoring of the presence of borreliae in Ixodes ricinus ticks in Brno park Pisárky, Czech Republic. Biológia.55;6: 657-662.
®ákovská A., 2000 b: Seasonal distribution of borreliae in Ixodes ricinus ticks in Brno park Pisárky. Scripta Fac Sci Nat Univ Masaryk Brun.26:33-42.
In the following articles the PCR as an used modern molecular techniques appeared
®ákovská, A., Čapková, L., Pejchalová, K., ©ikutová, S., Halouzka, J., Dendis, M., 2000: The study of presence of spirochetes focused on Borrelia burgdorferi sensu lato with PCR method in haematophagous and non haematophagous arthropods. Acta Parasitologica.45:p. 215
Janouąkovcová E., ®ákovská A., Halouzka J., Dendis M., 2002: Prevalence of Borrelia burgdorferi s. l. in Ixodes ricinus Ticks in localities of South Moravia and East Bohemia between 1996 - 2000 and Identification of isolated Species by PCR-RFLP and SDS-gradient PAGE Analysis. Scripta, Fac Sci Nat Univ Masaryk Brun.28 Suppl:41-50
®ákovská A., Baąta J., Horváth r., Dendis m., Pejchalová k., Hulínská D., 2002: Incidence of Borrelia burgdorferi sensu lato in Ixodes ricinus Ticks Using Dark-field Microscopy and Subsequent Single-step PCR. Skripta, Fac Sci Nat Univ Masaryk Brun.28. Suppl:3-9.
The last survey containing sequenation techniques was described in
Pejchalová, K., ®ákovská, A., Mejzlíková, M., Halouzka, J., Dendis, M., 2004: Isolation, Cultivation and Identification of Borrelia burgdorferi genospecies from Ixodes ricinus ticks from South Moravia, Czech Republic, Folia Microbiologica (Manuscript).
Mosquitoes
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.
Čapková L., ®ákovská A., Knoz J., Dendis M., ©erý O., 2002: Further spirochaetes findings in Culex sp., Aedes sp. mosquitoes and in Ixodes ricinus ticks. Biológia.57;3:389-394.
Čapková, L., Horváth, R., ®ákovská, A., 2002: Identification of spirochaete focused on Borrelia burgdorferi in mitgut of hematophagous and non hematophagous arthropods. Scripta Medica.75:p.43.
®ákovská A., Dendis M., Pejchalová K., 2000: Spirochaetes in Aedes species, Culex pipiens pipiens larvae and hibernating Culex pipiens molestus mosquitoes detected with dark field microscopy (DFM) and polymerase chain reaction (PCR) methods. Biológia.55;6:663-666.
®ákovská, A., Čapková, L., Holíková, A., ©erý, O., 2002: The presence of Borrelia burgdorferi in Culex (culex) pipiens larvae proved by the PCR Metod. Scripta, Fac Sci Nat Univ Masaryk Brun.28. Suppl:35-39.
Jörkova, M., Zakovska, A., Holikova, A.,2004: Summarized data of Borrelia burgdorferi s.l. presence in mosquito larva detected by PCR method in the Czech Republic. Ecological Question. 4:47-51.
®ákovská, A., Jörková, M., ©erý, O., Dendis, M, 2004.: Spirochaetes in Culex (C.) pipiens s. l. larvae. Biológia.59;2:283-287.
Small mites
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
Netuąil, J., ®ákovská, A., Horváth, R., Dendis, M., 2004: The presence of Borrelia burgdorferi sensu lato in mites parasiting at small rodents. Vector Borne and Zoonotic Dis. (Manuscript).
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
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).
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:
®ákovská, A., Nečasová, M., Dendis, M., Pechmann, V., 2000: The study of spirochete positivity focused on Borrelia burgdorferi in sera and heart rinses and tissues of wild ? living rodents. Acta Parasitologica. 45:p.218
®ákovská, A., Janouąkovcová, E., Dendis, M., Halouzka, J., 2002: Identification of isolated strains of Borrelia afzelii from rodent tissues using gradient PAGE and PCR methods. Skripta, Fac Sci Nat Univ Masaryk Brun.28 Suppl.:51-60.
Vostal, K., ®ákovská, A., 2003: Two year study of Examination of Blood from Wild-living Rodents for the Presence of Antiborrelian Antibodies. Ann Agric and Environ Med.10:1-4.
Raąková, G., ®ákovská, A., 2003: Comparation of immune response of different species of rodents after the antigenic stimulation of B.b.s.l. Physiol Res.52;3:pp38.
®ákovská, A., Janouąkovcová, A., Dendis, M., Halouzka J., 2004: Identification of 15 isolated strains of Borrelia afzelii from rodent tissues using PCR-RFLP and gradient PAGE method. Canadian Veterinary Journal. (Manuscript).
®ákovská, A., Vostal, K., Halouzka, J., 2004: Detection of the presence of antiborrelian antibodies and isolation of Borrelia afzelii from tissues of wild rodents. Immunol Lett.(Manuscript).
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Borrelia burgdorferi
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
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
®ákovská, A., Janouąkovcová, E., Halouzka, J., Dendis, M., 2000: Determination of spirochete isolates and studium of their protein composition with the PAGGE method. Acta Parasitologica.45:p. 213
Janouąkovcová, E., ®ákovská, A., Dendis M., 2002: Identifikace Ag struktur patogenních spirochet Borrelia burgdorferi a změny proteinového sloľení jednotlivých pasáľí metodou SDS-gradient PAGE. Scripta Medica.75:p. 40.
Identification of isolated strains with the characterisation of Flagellin gene and protein composition was the aim of the following articles
Janouąkovcová E., ®ákovská A., Halouzka J., Dendis M., 2002: Prevalence of Borrelia burgdorferi s. l. in Ixodes ricinus Ticks in localities of South Moravia and East Bohemia between 1996 - 2000 and Identification of isolated Species by PCR-RFLP and SDS-gradient PAGE Analysis. Scripta, Fac Sci Nat Univ Masaryk Brun.28 Suppl.:41-50.
Janouąkovcová, E., ®ákovská, A., Halouzka, J., Dendis, M., 2004: Occurrence of Borrelia afzelii and Borrelia garinii in Ixodes ricinus Ticks from Southern Moravia, Czech Republic. Vector Borne and Zoonotic Dis. 4;1:43-52
Study of immune response to B. afzelii antigen is involved in the artical
Helena Rusňáková, Alena ®ákovská, 2004: Borrelia afzelii and immune response. AAEM. (Manuscript)
The last survey containing sequenation technics focused on the flagellin gene of individual isolated strains was described in
Pejchalová, K., ®ákovská, A., Mejzlíková, M., Halouzka, J., Dendis, M., 2004: Isolation, Cultivation and Identification of Borrelia burgdorferi genospecies from Ixodes ricinus ticks from South Moravia, Czech Republic, Folia Microbiologica (Manuscript).
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