Two mice models for transferability of zoonotic bacteria via tick vector.

Spotted fever and typhus-related diseases caused by rickettsiae, Lyme borreliosis induced by spirochetes from Borrelia burgdorferii sensu lato complex, and Q fever evoked by Coxiella burnetii, are important zoonoses occurring worldwide. In order to study the pathogenesis of these infections, the efficacy of vaccines from the perspective of protection against the pathogens, pathogen - pathogen interactions during co-infections or pathogen-vector-host interrelationship, a suitable animal model should be established. In this study, we evaluated two mouse models - the C3H/N and Balb/c strains for susceptibility to infection and ability to transmit the pathogens via tick vector and to reveal the potential interactions between various bacterial tick-borne agents. Our results indicated that the C3H/N and Balb/c mice are well-accepted models of B. afzelii infection. However, they are not suitable for interaction studies with R. helvetica since the animals did not acquire rickettsiemia and do not transmit Rickettsia sp. to feeding ticks.

Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi.

BACKGROUND: Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology. RESULTS: Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids. CONCLUSION: Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato.

PURPOSE: For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. METHODS: Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. RESULTS: The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. CONCLUSION: The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

[Lyme borreliosis and demyelinating disease of the central nervous system]. / Lymská borelióza a demyelinizacné ochorenie CNS.

A case report is presented of a 55-year-old patient diagnosed with a demyelinating disease of unclear etiology. The patient had Lyme borreliosis in 2004. Specific IgG antibodies against B. burgdorferi s. l. were detected in the serum. Intrathecal antibodies were not found in the cerebrospinal fluid, but the presence of B. garinii DNA was confirmed by PCR analysis. It can be hypothesized that the borrelial persistence in the body may have been one of the triggers of the autoimmune process resulting in demyelination of the central nervous system (CNS).

Serological and molecular detection of Borrelia burgdorferi sensu lato and Anaplasmataceae in rodents.

The presence of Anaplasma spp. and Borrelia burgdorferi sensu lato in rodents from Eastern Slovakia were followed by serological and molecular methods. The seroprevalence for Borrelia was detected in 16.6 %, for Anaplasmataceae (APT) in 13.2 % and co-occurrence of Borrelia and APT in 7.5 %. Out of 110 ear biopsies of rodents, 5 were B. afzelii-positive. Five biopsies tested positive with the Ehr521-Ehr747 primers amplifying all the members of the family APT. A. phagocytophilum was detected in 1.8 %, 2.7 % were infected with Anaplasma-like organisms. Co-occurrence of Borrelia and Anaplasma in ear biopsies was found in 1.8 %. The circulation of both Borrelia and Anaplasma in the region of Eastern Slovakia was confirmed.

Serological evidence for Borrelia burgdorferi infection associated with clinical signs in dairy cattle in Slovakia.

In the course of epizootological research on Lyme borreliosis in domestic and farm animals, the serological evidence for the occurrence of this zoonosis in cattle was screened. An ELISA showed that 25.2% of cattle from seven geographical areas in Slovakia were positive for anti-Borrelia IgG antibodies. In particular localities, the seroprevalence ranged from 0.6% to 34.3%. Of 33 cases with clinical signs, 20 ELISA-positive samples were also confirmed in Western blots. The most frequent clinical signs were lameness and swollen joints, but most of the cases were asymptomatic. The occurrence of Borrelia burgdorferi antibodies and suspected clinical signs in cattle of Slovak regions indicates that veterinarians should pay attention to this disease in their clinical practice and include it within the differential diagnosis.