Spirochaetes and pathogenic Borrelia burgdorferi s.l. in mosquitoes (larvae and adults) in the Czech Republic

Alena Zakovska (1), Monika Jörkova (1), Jiri Halouzka (2), Milos Dendis (3)

1. Department of Comparative Animal Physiology and General Zoology, Faculty of Science, Masaryk University
2. Institute of Vertebrate Biology, Department of Medicine Zoology, Academy of Sciences, Klášterní 2, 691 42 Valtice, Czech Republic
3. Genetic Laboratory CKTCH, Výstavní 8, CZ-60200 Brno, Czech Republic

The aim of our study was the observation of the presence of spirochaetes, esp. Borrelia burgdorferi sensu lato in mosquitoes larvae and adults. As the question still remains if other hematophagous arthropods than ixodid ticks could participate in the enzootic cycle of Borrelia burgdorferi s.l. in nature. Adult mosquitoes were collected in 4 localities, mosquito larvae were collected in 3 localities in the Czech Republic. During the years 1997 – 2001 the presence of spirochaetes was observed in the midgut of 2 600 mosquitoe adults and 1798 larvae by dark field microscopy (DFM). Of all examined samples, 90 mosquito adults (3,5%) and 27 larvae (1,5 %) were positive for the presence of spirochaetes . Nighteen samples out of 90 adults (0,7%) and 10 samples out of 27 larvae (0,6 %) of all DFM positive exemplars were found to be positive for the presence of Borrelia burgdorferi s.l. DNA was detected by using two methods: the polymerase chain reaction (PCR) and PCR – RFLP. Nighteen PCR positive mosquitoe adults belonged to the following species : Culex (C.) pipiens s.l, Aedes (Ochlerotatus) communis s.l., Aedes (Aedes) cinereus, Aedes sp., 10 positive mosquito larvae were of Culex (C.) pipiens s.l., Culex (C.) pipiens pipiens mosquitoe species. By using SDS-gradient PAGE and PCR – RFLP methods the only isolated strain of mosquito adults was detected as Borrelia afzelii and one sample of mosquito larvae was detected by PCR-RFLP as Borrelia garinii. One of DFM positive sample from mosquitoe larvae was followly successfully isolated. According to PCR, PCR-RFLP, and gradient PAGE Metod this spirochaete did not Beliny to B. afzelii, B. garinii, B. burgdorferi sensu stricto. This spirochaete strain will be undertaken to further DNA detection. Results: Mosquitoes appeared to be positive for the presence of spirochaetes, but a very small number of them were revealed to be B. burgdorferi s. l. The question if they could be transferred into the Lyme borreliosis hosts remains open other experiments. This work was partially supported of CEZ: J 07/98: 143100008, 143100010, FRVS 541/03

The aim of our study was to monitore the positivity of Ixodes ricinus ticks for the presence of pathogenic Borrelia burgdorferi s.l. collected in the town park used as a recreation place for one hour at regular intervals of 14 days in the term of 6 years. Results from used methods such as DFM, PCR, PCR-RFLP, gradient PAGE were compared or complemented. Ixodes ricinus ticks occurred in each month of the year from April to October except November, December, January and February. A total of 3.732 Ixodes ricinus ticks were collected by flagging vegetation of a mixed forest town-park in Brno (350.000 inhabitants in the Czech Republic) from September 1996 to October 2002 and examined for the presence of spirochaetes by darkfield microscopy and from the year of 1998 for the presence of B.b. s.l. also using PCR method. 10 isolated strains were further identified by PCR-RFLP methods. Mean annual proportion of infected ticks was 6,9%; in 1996 6,3%, 1997 9,6%; 1998 12,4%; 1999 3,6%; 2000 4,1%; 2001 5,0%; 2002 12,3%. Mean annual proportion for all years was in larvae 2,7%; in nymphs 6,0%; in females 8,5%; in males 12,3%; Mean annual positivity of samples examined by PCR method was 5,0%; in larvae 1,0%; in nymphs 6,0%; in females 7,5%; in males 7,9%. The highest risk of infection with tick – borne borreliae for vertebrates (including man) occurred in the study area twice a year: the firs time in May-June, the second in September according to the number of positived ticks (the highest positivity was 20,5%). Using just cultivating method (2002) mean proportion appeared to be 11,8,%. 10 spirochaete isolated strains were obtained from positive ticks, and these were further determined by PCR-RFLP and by SDS- gradient PAGE method with the comparison of strain protein composition to standard stems (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae). 8 isolated strains were determined as to be Borrelia afzelii, 2 as Borrelia garinii. Other results will be prepared in tables and graphs. This work was partially supported of CEZ: J 07/98: 143100010, FRVS 541/03

The purpose of the study was to find the degree of antibody response on infection of Lyme disease caused by the pathogenic spirochaete Borrelia burgdorferi s.l. and the eventual presence of this bacteria in tissues of affected individuals (small rodents). A total of 450 wild-living rodents was trapped in locality Studenka in the North Moravia from the years of 1997-99 (276) to 2001-02 (174). All of these individuals were tested for the presence of antibodies against Borrelia burgdorferi sensu lato using indirect ELISA method. Serology was performed by testing for IgG antibodies against antigen of B. afzelii as one of the most presented genospecies of B.b. s.l. in wild small mammals in the Czech Republic. The results were sorted by period of trapping and species of examined rodents. 1. In the years of 1997-99 the positivity of trapped rodents was 36,4 %, the most positive species was Apodemus flavicollis (43,6 %). 2. In the years of 2001-02 the positivity of individuals was 43,8 %, the most positive species was Apodemus sylvaticus (58,8 %). For the presence of living borreliae in rodents a total of 219 rodents was also trapped in locality Studenka in the year of 1999. These individuals were examined for the presence of Libiny spirochaetes in rodent tissues by dark.field microscopy. Of all 39 samples were find to be positive (17,8 %) for the presence of spirochetes and 15 of them were successfully isolated. All isolated strains were identified as B. afzelii using PCR-RFLP method. The results were confirmed by usány SDS-PAGGE method. These data shows that rodents play the important role in the circulation of borreliae in nature.

This work was partially supported of CEZ: J 07/98: 143100008

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Zakovska A et al.: Spirochaetes and pathogenic Borrelia burgdorferi s.l. in mosquitoes (larvae and adults) in the Czech Republic