Andrias Hojgaard

Multilocus sequence analysis of Borrelia bissettii strains from North America reveals a new Borrelia species, Borrelia kurtenbachii.

Using multilocus sequence analyses (MLSA), we investigated the phylogenetic relationship of spirochaete strains from North America previously assigned to the genospecies Borrelia bissettii. We amplified internal fragments of 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) located on the main linear chromosome by polymerase chain reaction. Phylogenetic analysis of concatenated sequences of the 8 loci showed that the B. bissettii clade consisted of 4 closely related clusters which included strains from California (including the type strain DN127-Cl9-2/p7) and Colorado that were isolated from Ixodes pacificus, I. spinipalpis, or infected reservoir hosts. Several strains isolated from I. scapularis clustered distantly from B. bissettii. Genetic distance analyses confirmed that these strains are more distant to B. bissettii than they are to B. carolinensis, a recently described Borrelia species, which suggests that they constitute a new Borrelia genospecies. We propose that it be named Borrelia kurtenbachii sp. nov. in honour of the late Klaus Kurtenbach. The data suggest that ecological differences between B. bissettii and the new Borrelia genospecies reflect different transmission cycles. In view of these findings, the distinct vertebrate host-tick vector associations and the distributions of B. bissettii and B. kurtenbachii require further investigation.

Transmission dynamics of Borrelia burgdorferi s.s. during the key third day of feeding by nymphal Ixodes scapularis (Acari: Ixodidae).

Abstract Nymphal Ixodes scapularis Say are the principal vectors of Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) in the eastern United States. Physicians frequently face the decision of whether or not to administer prophylactic antibiotics to human tick bite victims in Lyme disease endemic regions, based on the overall probability that such bites will result in infection with B. burgdorferi s.s. We evaluated the transmission dynamics of B. burgdorferi s.s. during the key third day of nymphal I. scapularis feeding, when the risk of transmission rapidly increases. The cumulative probability that 50% of infected ticks transmitted B. burgdorferi s.s. occurred at 68 h of tick attachment and our overall estimate that a human tick bite would result in transmission of B. burgdorferi s.s. was 2.4%.

Coxiella Symbionts in the Cayenne Tick Amblyomma cajennense

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.

Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays

We have developed 2 real-time multiplex PCR assays for detection of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. The efficiency and sensitivity of each multiplex PCR assay was evaluated using field-collected Ixodes scapularis ticks that were positive for each of the pathogens, cloned plasmids harboring each of the PCR targets, and laboratory I. scapularis infected with B. burgdorferi B31. There was no difference in efficiency or sensitivity when comparing the multiplex PCR with the individual PCR reactions. If the 2 multiplex PCR assays are used in the same analysis, field-collected ticks that only harbor B. miyamotoi can also be identified. The multiplex assays are fast and cost-effective methods for screening and detecting pathogens in ticks, when compared to single-target PCR.

Vector competence of the blacklegged tick, Ixodes scapularis, for the recently recognized Lyme borreliosis spirochete Candidatus Borrelia mayonii

A novel species within the Borrelia burgdorferi sensu lato complex, provisionally named Borrelia mayonii, was recently found to be associated with Lyme borreliosis in the Upper Midwest of the United States. Moreover, B. mayonii was detected from host-seeking Ixodes scapularis, the primary vector of B. burgdorferi sensu stricto in the eastern United States. We therefore conducted a study to confirm the experimental vector competence of I. scapularis for B. mayonii (strain MN14-1420), using colony ticks originating from adults collected in Connecticut and CD-1 white mice. Larvae fed on mice 10 weeks after needle-inoculation with B. mayonii acquired spirochetes and maintained infection through the nymphal stage at an average rate of 12.9%. In a transmission experiment, 40% of naïve mice exposed to a single infected nymph developed viable infections, as compared with 87% of mice fed upon by 2-3 infected nymphs. Transmission of B. mayonii by one or more feeding infected nymphs was uncommon up to 48h after attachment (one of six mice developed viable infection) but occurred frequently when nymphs were allowed to remain attached for 72-96h or feed to completion (11 of 16 mice developed viable infection). Mice infected via tick bite maintained viable infection with B. mayonii, as determined by ear biopsy culture, for at least 28 weeks. Our results demonstrate that I. scapularis is capable of serving as a vector of B. mayonii. This finding, together with data showing that field-collected I. scapularis are infected with B. mayonii, indicate that I. scapularis likely is a primary vector to humans of this recently recognized Lyme borreliosis spirochete.

Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in Rodent Reservoirs and Ixodes scapularis Ticks Using a Doxycycline Hyclate-Laden Bait

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.

Molecular Identification of Salp15, a Key Salivary Gland Protein in the Transmission of Lyme Disease Spirochetes, From Ixodes persulcatus and Ixodes pacificus (Acari: Ixodidae)

ABSTRACT Salp15 is a multifunctional protein, vital to the tick in its need to obtain vertebrate host blood without stimulating a host inflammatory and immune response. The Salp15 protein from both Ixodes scapularis Say and Ixodes ricinus (L.), the principal vectors of the Lyme disease spirochete in eastern North America and Europe, respectively, have been well characterized and found to bind the murine CD4 receptor, DC-SIGN, and the OspC protein of Borrelia burgdorferi. In the current study, we characterized the full salp15 gene in Ixodes pacificus Cooley & Kohls and Ixodes persulcatus Schulze, the principal vectors of Lyme disease spirochetes in western North America and Asia, respectively. In comparing the Salp15 protein of all four principal vector ticks of public health importance for the transmission of Lyme disease spirochetes, we find the 53 C-terminal amino acids to have a high degree of similarity. There are at least three clades in the tree of Salp15 and its homologues, probably representing a multigene family.

Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov. prevail in diverse enzootic transmission cycles

Two species of the genus Borrelia, Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov., were first described by Postic and co-workers on the basis of genetic analyses of several loci. Multilocus sequence analysis of eight housekeeping loci confirmed that these two Borrelia genomospecies are distinct members of the Borrelia burgdorferi sensu lato complex. B. bissettiae sp. nov. was initially described in transmission cycles involving Neotoma fuscipes wood rats and Ixodes pacificus ticks in California, and Neotoma mexicana and Ixodes spinipalpis in Colorado. The preferred host of B. californiensis sp. nov. appears to be the California kangaroo rat, Dipodomys californicus; Ixodes jellisoni, I. spinipalipis and I. pacificus ticks are naturally infected with it. Thus, the ecological associations of the two genomospecies and their genetic distance from all other known Borrelia genomospecies species justify their description as separate genomospecies: B. bissettiae sp. nov. (type strain DN127T = DSM 17990T =  CIP 109136T) and B. californiensis (type strain CA446T = DSM 17989T = ATCC BAA-2689T).

Transmission of Borrelia miyamotoi sensu lato relapsing fever group spirochetes in relation to duration of attachment by Ixodes scapularis nymphs

Borrelia miyamotoi sensu lato relapsing fever group spirochetes are emerging as causative agents of human illness (Borrelia miyamotoi disease) in the United States. Host-seeking Ixodes scapularis ticks are naturally infected with these spirochetes in the eastern United States and experimentally capable of transmitting B. miyamotoi. However, the duration of time required from tick attachment to spirochete transmission has yet to be determined. We therefore conducted a study to assess spirochete transmission by single transovarially infected I. scapularis nymphs to outbred white mice at three time points post-attachment (24, 48, and 72h) and for a complete feed (>72-96h). Based on detection of B. miyamotoi DNA from the blood of mice fed on by an infected nymph, the probability of spirochete transmission increased from 10% by 24h of attachment (evidence of infection in 3/30 mice) to 31% by 48h (11/35 mice), 63% by 72h (22/35 mice), and 73% for a complete feed (22/30 mice). We conclude that (i) single I. scapularis nymphs effectively transmit B. miyamotoi relapsing fever group spirochetes while feeding, (ii) transmission can occur within the first 24h of nymphal attachment, and (iii) the probability of transmission increases with the duration of nymphal attachment.

Molecular Characterization of a New Isolate of Borrelia lusitaniae Derived from Apodemus sylvaticus in Portugal

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.