Infection Kinetics and Tropism of Borrelia burgdorferi sensu lato in Mouse After Natural (via Ticks) or Artificial (Needle) Infection Depends on the Bacterial Strain

Abstract

Borrelia burgdorferi sl is a complex of pathogen bacteria transmitted to the host by Ixodes ticks. European Ixodes ricinus ticks transmit different B. burgdorferi species, pathogenic to human. Bacteria are principally present in unfed tick midgut, then migrate to salivary glands during blood meal and infect a new host via saliva. In this study, efficiency of transmission in a mouse model of three pathogen species belonging to the B. burgdorferi sl complex, B. burgdorferi sensu stricto (B31, N40, and BRE-13), B. afzelii (IBS-5), and B. bavariensis (PBi) is examined in order to evaluate infection risk after tick bite. We compared the dissemination of the Borrelia species in mice after tick bite and needle injection. Location in the ticks and transmission to mice were also determined for the three species by following infection kinetics. After inoculation, we found a significant prevalence in the brain for PBi and BRE-13, in the heart, for PBi, in the skin where B31 was more prevalent than PBi and in the ankle where both B31 and N40 were more present than PBi. After tick bite, statistical analyses showed that BRE-13 was more prevalent than N40 in the brain, in the bladder and in the inguinal lymph node. When Borrelia dissemination was compared after inoculation and tick bite, we observed heart infection only after tick inoculation of BRE-13, and PBi was only detected after tick bite in the skin. For N40, a higher number of positive organs was found after inoculation compared to tick bite. All European B. burgdorferi sl strains studied were detected in female salivary glands before blood meal and infected mice within 24 h of tick bite. Moreover, Borrelia-infected nymphs were able to infect mice as early as 12 h of tick attachment. Our study shows the need to remove ticks as early as possible after attachment. Moreover, Borrelia tropism varied according to the strain as well as between ticks bite and needle inoculation, confirming the association between some strains and clinical manifestation of Lyme borreliosis, as well as the role played by tick saliva in the efficiency of Borrelia infection and dissemination in vertebrates.

Keywords: Borrelia; Ixodes ricinus; lyme borreliosis; mouse; tick bite; transmission.

Figure 1

Data acquisition workflow. (A) Mice were infected by subcutaneous inoculation of 10⁵ bacteria. Their infection status was controlled by PCR after ear biopsy. Borrelia-positive mice were either used for infecting larvae or nymphs (B,C respectively) or dissected 38 days after infection; B and C. Borrelia-negative larvae or nymphs were fed on Borrelia-positive mice. After molting, their rate of infection was determined; (D) Ten Borrelia-exposed nymphs or 3 Borrelia-exposed females were fed on Borrelia-negative mice. The kinetics of infection of their salivary glands was determined after different duration of tick attachment; (E) Mice exposed to the bite of Borrelia-exposed nymphs or female ticks for different durations of tick attachment were dissected 38 days after tick bite.

Figure 2

Detection of Borrelia afzelii IBS-5 by nested PCR in the organs of mice after needle inoculation. Mice were infected by subcutaneous inoculation of 10⁵ bacteria. Organs were dissected at 38 days post-infection. DNA was extracted as described in the Methods. A first step of PCR was performed followed by a second step of nested PCR. Negative controls (PCR mix) and positive controls (DNA extracted from a culture of Borrelia burgdorferi sl bacteria) were used on each gel. (A) Organs of mice infected by B. afzelii IBS-5. The positive control was a DNA extracted from B. garinii 20047 strain. (B) Organs of a non-infected mouse. Two positive controls were used. Positive control n°1: B. garinii 20047 strain amplified in the first PCR. Positive control 2: B. garinii 20047 strain; Negative control: PCR mix.