Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1.

Borrelia burgdorferi, the agent of Lyme borreliosis, can elude hosts' innate and adaptive immunity as part of the course of infection. The ability of B. burgdorferi to invade or be internalized by host cells in vitro has been proposed as a mechanism for the pathogen to evade immune responses or antimicrobials. We have previously shown that B. burgdorferi can be internalized by human neuroglial cells. In this study we demonstrate that these cells take up B. burgdorferi via coiling phagocytosis mediated by the formin, Daam1, a process similarly described for human macrophages. Following coincubation with glial cells, B. burgdorferi was enwrapped by Daam1-enriched coiling pseudopods. Coiling of B. burgdorferi was significantly reduced when neuroglial cells were pretreated with anti-Daam1 antibody indicating the requirement for Daam1 for borrelial phagocytosis. Confocal microscopy showed Daam1 colocalizing to the B. burgdorferi surface suggesting interaction with borrelial membrane protein(s). Using the yeast 2-hybrid system for identifying protein-protein binding, we found that the B. burgdorferi surface lipoprotein, BBA66, bound the FH2 subunit domain of Daam1. Recombinant proteins were used to validate binding by ELISA, pull-down, and co-immunoprecipitation. Evidence for native Daam1 and BBA66 interaction was suggested by colocalization of the proteins in the course of borrelial capture by the Daam1-enriched pseudopodia. Additionally, we found a striking reduction in coiling for a BBA66-deficient mutant strain compared to BBA66-expressing strains. These results show that coiling phagocytosis is a mechanism for borrelial internalization by neuroglial cells mediated by Daam1.

Effects of low-temperature flea maintenance on the transmission of Yersinia pestis by Oropsylla montana.

Yersinia pestis, the causative agent of plague, is primarily a rodent-associated, flea-borne zoonosis maintained in sylvatic foci throughout western North America. Transmission to humans is mediated most commonly by the flea vector Oropsylla montana and occurs predominantly in the southwestern United States. With few exceptions, previous studies showed O. montana to be an inefficient vector at transmitting Y. pestis at ambient temperatures, particularly when such fleas were fed on susceptible hosts more than a few days after ingesting an infectious blood meal. We examined whether holding fleas at subambient temperatures affected the transmissibility of Y. pestis by this vector. An infectious blood meal containing a virulent Y. pestis strain (CO96-3188) was given to colony-reared O. montana fleas. Potentially infected fleas were maintained at different temperatures (6°C, 10°C, 15°C, or 23°C). Transmission efficiencies were tested by allowing up to 15 infectious fleas to feed on each of 7 naïve CD-1 mice on days 1-4, 7, 10, 14, 17, and 21 postinfection (p.i.). Mice were monitored for signs of infection for 21 days after exposure to infectious fleas. Fleas held at 6°C, 10°C, and 15°C were able to effectively transmit at every time point p.i. The percentage of transmission to naïve mice by fleas maintained at low temperatures (46.0% at 6°C, 71.4% at 10°C, 66.7% at 15°C) was higher than for fleas maintained at 23°C (25.4%) and indicates that O. montana fleas efficiently transmit Y. pestis at low temperatures. Moreover, pooled percent per flea transmission efficiencies for flea cohorts maintained at temperatures of 10°C and 15°C (8.67% and 7.87%, respectively) showed a statistically significant difference in the pooled percent per flea transmission efficiency from fleas maintained at 23°C (1.94%). This is the first comprehensive study to demonstrate efficient transmission of Y. pestis by O. montana fleas maintained at temperatures as low as 6°C. Our findings further contribute to the understanding of plague ecology in temperate climates by providing support for the hypothesis that Y. pestis is able to overwinter within the flea gut and potentially cause infection during the following transmission season. The findings also might hold implications for explaining the focality of plague in tropical regions.

Molecular ecology of Listeria monocytogenes and other Listeria species in small and very small ready-to-eat meat processing plants.

A longitudinal study was conducted to track Listeria contamination patterns in ready-to-eat meats from six small or very small meat processing plants located in three states over 1 year. A total of 688 environmental sponge samples were collected from nonfood contact surfaces during bimonthly visits to each plant. Overall, L. monocytogenes was isolated from 42 (6.1%) environmental samples, and its prevalence ranged from 1.7 to 10.8% across different plants. Listeria spp., other than L. monocytogenes, were isolated from 9.5% of samples overall, with the prevalence ranging from 1.5 to 18.3% across different plants. The prevalence of L. monocytogenes correlated well with that of other Listeria spp. for some but not all plants. One L. monocytogenes isolate representing each positive sample was characterized by molecular serotyping, EcoRI ribotyping, and pulsed-field gel electrophoresis typing. Seven sample sites tested positive for L. monocytogenes on more than one occasion, and the same ribotype was detected more than once at five of these sites. Partial sigB sequencing was used to speciate other Listeria spp. isolates and assign an allelic type to each isolate. Other Listeria spp. were isolated more than once from 14 sample sites, and the same sigB allelic type was recovered at least twice from seven of these sites. One plant was colonized by an atypical hemolytic L. innocua strain. Our findings indicate that small and very small meat processing plants that produce ready-to-eat meat products are characterized by a varied prevalence of Listeria, inconsistent correlation between contamination by L. monocytogenes and other Listeria spp., and a unique Listeria molecular ecology.

Effects of temperature on the transmission of Yersinia Pestis by the flea, Xenopsylla Cheopis, in the late phase period.

BACKGROUND: Traditionally, efficient flea-borne transmission of Yersinia pestis, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of Xenopsylla cheopis with a wild type strain of Y. pestis and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using Y. pestis-specific antigen or antibody detection assays on mouse tissues. RESULTS: Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted Y. pestis infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures. CONCLUSIONS: Our results suggest that temperature does not significantly effect the per flea efficiency of Y. pestis transmission by X. cheopis, but that temperature is likely to influence the dynamics of Y. pestis flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether Y. pestis biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.