Longitudinal study of prevalence and spatio-temporal distribution of Borrelia burgdorferi sensu lato in ticks from three defined habitats in Latvia, 1999-2010.

Members of the Borrelia burgdorferi sensu lato (s.l.) species complex are known to cause human Lyme borreliosis. Because of longevity of some reservoir hosts and the Ixodes tick vectors' life cycle, long-term studies are required to better understand species and population dynamics of these bacteria in their natural habitats. Ticks were collected between 1999 and 2010 in three ecologically different habitats in Latvia. We used multilocus sequence typing utilizing eight chromosomally located housekeeping genes to obtain information about species and population fluctuations and/or stability of B. burgdorferi s.l. in these habitats. The average prevalence over all years was 18.9%. From initial high-infection prevalences of 25.5%, 33.1% and 31.8%, from 2002 onwards the infection rates steadily decreased to 7.3%. Borrelia afzelii and Borrelia garinii were the most commonly found genospecies but striking local differences were obvious. In one habitat, a significant shift from rodent-associated to bird-associated Borrelia species was noted whilst in the other habitats, Borrelia species composition was relatively stable over time. Sequence types (STs) showed a random spatial and temporal distribution. These results demonstrated that there are temporal regional changes and extrapolations from one habitat to the next are not possible.

Comparison of methods for economic and efficient tick and Borrelia DNA purification.

DNA purification is a critical step in the processing of samples for molecular diagnosis and/ or identification of pathogens via polymerase chain reaction (PCR). Especially when handling vectors like ticks, purifying the DNA always poses a challenge. In this study, we compared factors that may have an influence on DNA extraction namely commercially available DNA extraction kits vs alkaline hydrolysis for DNA extraction. The methods were applied to questing Ixodes (I.) ricinus ticks and Borrelia cultures of defined cell concentrations. A total of 69 questing I. ricinus ticks were collected. From 34 ticks, total DNA was extracted using a commercial DNA extraction kit. Thirty-five ticks were treated with 1.25% ammonium hydroxide (NH4OH). Six ticks from each batch were placed in 70% ethanol (EtOH) for one week prior to DNA extraction to see the effect of EtOH preservation on total DNA yield. DNA yield was estimated in field-collected ticks using conventional PCR targeting the Ixodes Cytochrome C oxidase (coi) gene and in cultured Borrelia isolates using quantitative real-time PCR (qPCR) targeting the FlaB encoding gene of Borrelia. Column DNA extraction yielded slightly better results than NH4OH treatment when tested in a PCR targeting a tick-specific coi gene (96% PCR-positive vs 86% PCR-positive results, respectively). EtOH preservation had a slightly negative effect on DNA yield and - again - slightly stronger PCR products were observed by commercial kit extraction. A Shapiro-Wilk test conducted revealed a significance-level of 90% for both the methods, indicating a normal distribution of the values generated by BioNumerics quantification. A two-sided t-test conducted revealed a significant (p < 0.01) mean difference between the methods. Similarly, qPCR on cultured specimen DNA of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s.) (B31) with different concentrations revealed a better yield for kit extraction in comparison to NH4OH treatment; a difference of approximately 3 Ct-values was ascertained between extraction methods. A one-sided t-test showed a significant difference between the methods at lower concentration of Borrelia i.e. better extraction with a commercial kit at lower borrelial DNA concentration, while at higher concentration (106 cells per ml) the difference was not significant.