Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.

INTRODUCTION: Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tick-borne pathogens (TBP) is mainly by molecular techniques based on polymerase chain reactions (PCR). OBJECTIVE: To design and evaluate a multiplex PCR for the molecular screening of zoonotic TBP for exploratory studies. MATERIAL AND METHODS: Control DNA from reference strains, DNA from experimentally-infected biological specimens, and from Rhipicephalus sanguineus ticks collected from domestic and homeless dogs were used. A multiplex PCR assay to detect the presence of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. was designed and optimized using primers previously reported for B. burgdorferi sensu lato and Anaplasma spp., while for Babesia spp. they were designed in silico. The multiplex PCR was evaluated on the DNA from biological samples. RESULTS: A new set of specific primers for Babesia spp. was designed. Adjustment of the master mix reactive concentrations and amplification conditions for the multiplex PCR allowed the successful amplification of the specific amplicons for each microbial group from the control DNA and experimentally-infected biological specimens. The efficiency of the multiplex PCR amplifying three DNA targets was confirmed. Individual and co-infection of Anaplasma spp. and Babesia spp. were detected in the R. sanguineus ticks from dogs. CONCLUSIONS: A multiplex PCR assay for the screening of three TBP is available. By using it, B. burgdorferi sensu lato, Anaplasma spp. and Babesia spp. can be detected accurately in one PCR reaction.

The high prevalence and diversity of Chlamydiales DNA within Ixodes ricinus ticks suggest a role for ticks as reservoirs and vectors of Chlamydia-related bacteria.

The Chlamydiales order is composed of nine families of strictly intracellular bacteria. Among them, Chlamydia trachomatis, C. pneumoniae, and C. psittaci are established human pathogens, whereas Waddlia chondrophila and Parachlamydia acanthamoebae have emerged as new pathogens in humans. However, despite their medical importance, their biodiversity and ecology remain to be studied. Even if arthropods and, particularly, ticks are well known to be vectors of numerous infectious agents such as viruses and bacteria, virtually nothing is known about ticks and chlamydia. This study investigated the prevalence of Chlamydiae in ticks. Specifically, 62,889 Ixodes ricinus ticks, consolidated into 8,534 pools, were sampled in 172 collection sites throughout Switzerland and were investigated using pan-Chlamydiales quantitative PCR (qPCR) for the presence of Chlamydiales DNA. Among the pools, 543 (6.4%) gave positive results and the estimated prevalence in individual ticks was 0.89%. Among those pools with positive results, we obtained 16S rRNA sequences for 359 samples, allowing classification of Chlamydiales DNA at the family level. A high level of biodiversity was observed, since six of the nine families belonging to the Chlamydiales order were detected. Those most common were Parachlamydiaceae (33.1%) and Rhabdochlamydiaceae (29.2%). "Unclassified Chlamydiales" (31.8%) were also often detected. Thanks to the huge amount of Chlamydiales DNA recovered from ticks, this report opens up new perspectives on further work focusing on whole-genome sequencing to increase our knowledge about Chlamydiales biodiversity. This report of an epidemiological study also demonstrates the presence of Chlamydia-related bacteria within Ixodes ricinus ticks and suggests a role for ticks in the transmission of and as a reservoir for these emerging pathogenic Chlamydia-related bacteria.

Serological evidence of tick-borne encephalitis virus infection in rodents captured at four sites in Switzerland.

In a previous study, the presence of tick-borne encephalitis virus (TBEV) in questing Ixodes ricinus L. ticks and in field derived ticks that engorged on small mammals (n = 9,986) was investigated at four sites located in a TBE area in Switzerland. Two of these sites were already recognized as TBE foci (Thun and Belp) and the screening of ticks revealed the presence of TBEV in ticks at a third site, Kiesen, but not at the fourth one, Trimstein. The aim here was to test another approach to detect TBE endemic areas. Sera from 333 small mammals (Apodemus flavicollis, A. sylvaticus, Myodes glareolus) captured in 2006 and 2007 at the four sites were examined for the presence of antibodies against TBEV using immunofluorescence and avidity tests. Overall the prevalence of antibodies against TBEV in rodents reached 3.6% (12/333). At two sites known as TBE foci, Thun and Belp, anti-TBEV antibodies were detected in 9.9% (9/91) and 1.6% (1/63) of rodent sera, respectively. At the third site, Kiesen, recently identified as a TBE focus by the detection of TBEV in ticks, anti-TBEV antibodies were detected in 1.8% (2/113) of rodent sera. Finally, at Trimstein, none of the examined rodent sera had antibodies against TBEV (0/66). This study shows another approach to detect TBE foci by testing antibodies in small mammal sera that is less time-consuming and less expensive than molecular tools.

Pathogens of emerging tick-borne diseases, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp., in ixodes ticks collected from rodents at four sites in Switzerland (Canton of Bern).

This study is part of a project that aimed to better understand the role of small mammals in the maintenance of the tick-borne encephalitis virus at four different sites in Switzerland. Here we focused on the detection of three intracellular pathogens, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp., in field-derived ticks that detached from 79 small mammals. We analyzed 465 Ixodes ricinus larvae after their molt and 14 semiengorged I. trianguliceps that were feeding on rodents. No pathogen was detected in I. trianguliceps. In I. ricinus, the most frequently detected pathogen was Rickettsia spp. (7.3%). All Rickettsia spp. identified DNA belonged to R. helvetica except one DNA sample that was identified as R. monacensis. The prevalence of Babesia spp. reached 2.4% and identification at the species level revealed B. venatorum (1.7%) and B. microti (0.4%). A. phagocytophilum was not detected in I. ricinus that detached from rodents. To verify the absence of A. phagocytophilum at the four sites, additional questing nymphs collected at these sites were analyzed for A. phagocytophilum. This pathogen was detected at one site only, where 2% (2/100) of questing ticks were infected. Some of these emerging pathogens are described for the first time in molted larvae that fed on rodents. The presence of medically relevant pathogens, with a global prevalence of 9.9%, highlights the importance to inform the medical corporation on the risk for human health in these areas.

Variable spikes in tick-borne encephalitis incidence in 2006 independent of variable tick abundance but related to weather.

BACKGROUND: The incidence of tick-borne encephalitis showed a dramatic spike in several countries in Europe in 2006, a year that was unusually cold in winter but unusually warm and dry in summer and autumn. In this study we examine the possible causes of the sudden increase in disease: more abundant infected ticks and/or increased exposure due to human behaviour, both in response to the weather. METHODS: For eight countries across Europe, field data on tick abundance for 2005-2007, collected monthly from a total of 41 sites, were analysed in relation to total annual and seasonal TBE incidence and temperature and rainfall conditions. RESULTS: The weather in 2006-2007 was exceptional compared with the previous two decades, but neither the very cold start to 2006, nor the very hot period from summer 2006 to late spring 2007 had any consistent impact on tick abundance. Nor was the TBE spike in 2006 related to changes in tick abundance. Countries varied in the degree of TBE spike despite similar weather patterns, and also in the degree to which seasonal variation in TBE incidence matched seasonal tick activity. CONCLUSION: The data suggest that the TBE spike was not due to weather-induced variation in tick population dynamics. An alternative explanation, supported by qualitative reports and some data, involves human behavioural responses to weather favourable for outdoor recreational activities, including wild mushroom and berry harvest, differentially influenced by national cultural practices and economic constraints.

A comparison of two DNA extraction approaches in the detection of Borrelia burgdorferi sensu lato from live Ixodes ricinus ticks by PCR and reverse line blotting.

We tested two approaches to extract Borrelia DNA from live Ixodes ricinus ticks before polymerase chain reaction (PCR) and reverse line blotting (RLB): DNA extraction of one half of the tick after incubation in BSK medium and DNA extraction of the other half of the tick directly, using ammonium hydroxide. Among 2079 ticks, 31.2% (n=649) were found to be Borrelia-infected by PCR-RLB test using at least one of the DNA extraction methods. Five hundred four ticks (24.2%) were found infected after incubation in BSK and 481 (23.1%) after direct DNA extraction from the tick. The difference was not significant. However, these prevalences were significantly lower when only one method was applied (23.1% and 24.2%) compared to the prevalence obtained by the use of both methods (31.2%). In 313 infected ticks discordant results were obtained, i.e., one half of the tick was found to be infected whereas the other half was uninfected. Among these ticks, B. garinii and B. burgdorferi sensu stricto (ss) were significantly more frequently identified in the half tick incubated in BSK. No significant differences were observed for B. burgdorferi ss, B. valaisiana, and for undetermined Borrelia species.

Detection and identification of Borrelia burgdorferi sensu lato genospecies in ticks from three different regions in Slovakia.

Lyme borreliosis is one of the most common tick-borne diseases that occur in Slovakia. In this study, Borrelia burgdorferi sensu lato was detected and cultivated from questing ticks collected in three areas of Slovakia. Two methods, restriction fragment length polymorphism and reverse line blot, were used for identification of isolates and determination of the prevalence of B. burgdorferi s.l. in the ticks. The prevalence of B. burgdorferi s.l. in I. ricinus detected by reverse line blot was 31.9%. Four genospecies, namely B. garinii, B. valaisiana, B. afzelii and B. burgdorferi sensu stricto were found. B. garinii was the most prevalent genospecies.

Ixodes ricinus density and infection prevalence of Borrelia burgdorferi sensu lato along a North-facing altitudinal gradient in the Rhône Valley (Switzerland).

Questing Ixodes ricinus ticks were sampled monthly along a north-facing altitudinal gradient in the canton of Valais, Switzerland, from March 2004 to February 2005. Tick density and infection with Borrelia burgdorferi sensu lato were monitored. Ticks were collected by flagging vegetation at three different altitudes (750 m, 880 m, and 1020 m above sea level). Ticks were examined for Borrelia by polymerase chain reaction (PCR) followed by reverse line blot. At the three altitudes, questing tick activity was not observed under 10 degrees C and was reduced when saturation deficit was higher than 5 mm Hg, most questing tick activity was occurred between 2 mm Hg and 7 mm Hg. Tick density and peak tick density were highest at 1020 m. High saturation deficits at the lowest altitudes appear to impair the tick population. The prevalence of B. burgdorferi infection in nymphs and adults decreased with altitude. The prevalence of infection was higher in adult ticks (47%) than in nymphs (29%). Four B. burgdorferi sensu lato genospecies were detected: B. afzelii (40%), B. garinii (22%), B. valaisiana (12%) and B. burgdorferi sensu stricto (6%). Mixed infections were detected in 13% of infected ticks.