Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent

A physical map of the 952kbp chromosome of Borrelia burgdorferi Sh-2-82 has been constructed. Eighty-three intervals on the chromosome, defined by the cleavage sites of 15 restriction enzymes, are delineated. The intervals vary in size from 96kbp to a few hundred bp, with an average size of 11.5 kbp. A striking feature of the map is its linearity; no other bacterial groups are known to have linear chromosomes. The two ends of the chromosome do not hybridize with one another, indicating that there are no large common terminal regions. The chromosome of this strain was found to be stable in culture; passage 6, 165 and 320 cultures have identical chromosomal restriction maps. We have positioned all previously known Borrelia burgdorferi chromosomal genes and several newly identified ones on this map. These include the gyrA/gyrB/dnaA/dnaN gene cluster, the rRNA gene cluster, fla, flgE, groEL (hsp60), recA, the rho/hip cluster, the dnaK (hsp70)/dnaJ/grpE cluster, the pheT/pheS cluster, and the genes which encode the potent immunogen proteins p22A, p39 and p83. Our electrophoretic analysis detects five linear and at least two circular plasmids in B. burgdorferi Sh-2-82. We have constructed a physical map of the 53 kbp linear plasmid and located the operon that encodes the two major outer surface proteins ospA and ospB on this plasmid. Because of the absence of functional genetic tools for this organism, these maps will serve as a basis for future mapping, cloning and sequencing studies of B. burgdorferi.     

Emulsifier Dependent <Emphasis Type="Italic">in vitro</Emphasis> Digestion and Bioaccessibility of β-Carotene Loaded in Oil-in-Water Emulsions

This study was performed to examine the effect of emulsifiers used to coat emulsion droplets containing β-carotene on the behavior of lipid digestion and bioaccessibility. Different emulsifiers (whey protein isolate, soy protein isolate, sodium caseinate, Tween 20, and soy lecithin) were used to prepare emulsions with similar sized droplets (200–400 nm). Protein-stabilized emulsions showed a similar behavior of digestion, and morphological change in the simulated gastrointestinal conditions. Soy lecithin-stabilized emulsions showed the lowest rate and extent of lipid digestion probably due to the low emulsifying capability of soy lecithin, showing coalesced droplets occurring after exposure to the gastric phase. Tween 20-stabilized emulsions had a lower rate and extent of lipid digestion than that of protein-stabilized emulsions, even though Tween 20-stabilized emulsions had a more stable structure to resistant to aggregation in gastric phase. Even though the difference in the digestion rate and extent, β-carotene bioaccessibility was not significantly different among emulsions stabilized by different emulsifiers at p?<?0.05.     

Heat Shock Cognate Protein 71-Associated Peptides Function as an Epitope for Toxoplasma gondii-Specific CD4+ CTL

HLA-DR-restricted CD4⁺ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4⁺ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4⁺ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4⁺ CTL.     

Host-seeking behavior of Ixodes pacificus (Acari: Ixodidae) nymphs in relation to environmental parameters in dense-woodland and woodland-grass habitats

In the far-western United States, the bacteria that cause Lyme disease (Borrelia burgdorferi, Bb) and human granulocytic anaplasmosis (Anaplasma phagocytophilum, Ap) are transmitted by the western black-legged tick (Ixodes pacificus). In a dense woodland, human behaviors involving contact with wood were recently found to pose greater risk for encountering I. pacificus nymphs than behaviors entailing exclusive exposure to leaf litter. A four-year follow-up study was undertaken in the same woodland and, as a comparison area in one year only, in a nearby woodland-grass habitat to explore the biotic and abiotic factors that might elevate human exposure to host-seeking nymphs. Nymphs were active in the dense woodland throughout the daytime, but no consistent pattern of activity was observed with respect to time of day, temperature, or relative humidity. Significantly more nymphs were collected from the southern aspects of dense-woodland trunks than from other aspects, and more nymphs quested at a height of < or = 1 m vs 1-2 m aboveground. The prevalence of bacterial infection in ticks from the dense woodland was highly variable among years, with maxima of 22.6% and 42.9% for Bb, and 15.6% and 1.8% for Ap, in nymphs from logs and trunks, respectively. The mean densities of nymphs, and of Bb- or Ap-infected nymphs, were typically higher on logs and trunks than in adjoining leaf litter or grass in both habitats. The acarologic risk of encountering an infected nymph on dense-woodland logs or trunks was 2.8 to 11 times higher for Bb than for Ap in two of three years, and it was usually higher in dense woodland than in woodland-grass for both agents. Coinfections were rare (0.27%, n = 369 nymphs from both habitats). Individuals having prolonged contact with logs or trunks in spring would be well advised to employ personal protective measures to minimize exposure to I. pacificus nymphs and their attendant bacterial zoonotic agents.     

Rapid recovery of nuclear and mitochondrial genes by genome skimming from Northern Hemisphere freshwater crayfish

Molecular phylogenetics has benefited tremendously from the advent of next‐generation sequencing, enabling quick and cost‐effective recovery of whole mitogenomes via an approach referred to as ‘genome skimming’. Recently, genome skimming has been utilised to recover highly repetitive nuclear genes such as 18S and 28S ribosomal RNA genes that are useful for inferring deeper evolutionary relationships. To address some outstanding issues in the relationships among Northern Hemisphere freshwater crayfish (Astacoidea), we sequenced the partial genome of crayfish species from Asian, North American and European genera and report the successful recovery of whole mitogenome sequences in addition to three highly repetitive nuclear genes, namely histone H3, 18S and 28S ribosomal RNA. Consistent with some previous studies using short mtDNA and nuclear gene fragments, phylogenetic analyses based on the concatenation of recovered mitochondrial and/or nuclear sequences recovered the Asian cambarid lineage as basal to all astacids and North American cambarids, which conflicts with the current taxonomic classification based on morphological and reproduction‐related characters. Lastly, we show that complete H3, 18S and 28S ribosomal RNA genes can also be consistently recovered from a diverse range of animal taxa, demonstrating the potential wide utility of genome skimming for nuclear markers.     

Transmission cycles of Borrelia burgdorferi and B. bissettii in relation to habitat type in northwestern California

This study was undertaken to determine which rodent species serve as primary reservoirs for the Lyme disease spirochete Borrelia burgdorferi in commonly occurring woodland types in inland areas of northwestern California, and to examine whether chaparral or grassland serve as source habitats for dispersal of B. burgdorferi‐ or B. bissettii‐infected rodents into adjacent woodlands. The western gray squirrel (Sciurus griseus) was commonly infected with B. burgdorferi in oak woodlands, whereas examination of 30 dusky‐footed woodrats (Neotoma fuscipes) and 280 Peromyscus spp. mice from 13 widely‐spaced Mendocino County woodlands during 2002 and 2003 yielded only one infected woodrat and one infected deer mouse (P. maniculatus). These data suggest that western gray squirrels account for the majority of production by rodents of fed Ixodes pacificus larvae infected with B. burgdorferi in the woodlands sampled. Infections with B. burgdorferi also were rare in woodrats (0/47, 0/3) and mice (3/66, 1/6) captured in chaparral and grassland, respectively, and therefore these habitats are unlikely sources for dispersal of this spirochete into adjacent woodlands. On the other hand, B. bissettii was commonly detected in both woodrats (22/47) and mice (15/66) in chaparral. We conclude that the data from this and previous studies in northwestern California are suggestive of a pattern where inland oak‐woodland habitats harbor a B. burgdorferi transmission cycle driven primarily by I. pacificus and western gray squirrels, whereas chaparral habitats contain a B. bissettii transmission cycle perpetuated largely by I. spinipalpis, woodrats, and Peromyscus mice. The dominant role of western gray squirrels as reservoirs of B. burgdorferi in certain woodlands offers intriguing opportunities for preventing Lyme disease by targeting these animals by means of either host‐targeted acaricides or oral vaccination against B. burgdorferi.