Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.     

An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation

A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.     

Long-term changes in ecosystem health of two Hudson Valley watersheds, New York, USA, 1936–2001

We examined long-term ecological change in two Hudson River tributaries, the Wappinger and Fishkill Creek watersheds in Dutchess County, New York State. Fish data spanning 65 years (1936, 1988, 1992, and 2001) and shorter term macroinvertebrate data (1988, 2001) were used to assess the influence of land use practices. Between 1988 and 2001, macroinvertebrate index Biotic Assessment Profile (BAP) improved by 113–165% in the Fishkill Creek watershed, and fish Index of Biotic Integrity (IBI) improved by 117–140%. Fish IBI and fish species richness were significantly different (p < 0.01) between the watersheds, with Wappinger Creek in better condition. Long-term fish IBI scores showed degradation in both watersheds since the 1930s. Changes in species composition suggest community homogenization on par with overall changes in the fish fauna of New York. Most notable were increases in tolerant species and declines in intolerant or moderately tolerant species. Whereas Fishkill Creek IBIs showed decline in 1988 relative to 1936, followed by improvement, Wappinger Creek declined monotonically in environmental quality. Development has intensified in both watersheds, but Fishkill Creek is improving while Wappinger Creek watershed is undergoing less mitigated degradation. We find that older, semi-quantitative data can be used to construct environmental quality indicators, and can be of great use for measuring long-term change.     

A vegetable oil feeding history affects digestibility and intestinal fatty acid uptake in juvenile rainbow trout Oncorhynchus mykiss

Future expansion of aquaculture relies on the use of alternatives to fish oil in fish feed. This study examined to what extent the nature of the feed oil affects intestinal lipid uptake properties in rainbow trout. The fish were fed a diet containing fish (FO), rapeseed (RO) or linseed (LO) oil for 8 weeks after which absorptive properties were assessed. Differences in digestibility due to feed oil history were measured using diet FO with an indigestible marker. Intestinal integrity, paracellular permeability, in vitro transepithelial fatty acid transport (³H-18:3n − 3 and ¹⁴C-16:0) and their incorporation into intestinal epithelia were compared using Ussing chambers. Feed oil history did not affect the triacylglycerol/phosphatidylcholine ratio (TAG/PC) of the newly synthesized lipids in the segments. The lower TAG/PC ratio with 16:0 (2:1) than with 18:3 (10:1) showed the preferential incorporation of 16:0 into polar lipids. The FO-feeding history decreased permeability and increased transepithelial resistance of the intestinal segments. Transepithelial passage rates of 18:3n − 3 were higher when pre-fed LO compared to RO or FO. Similarly, pre-feeding LO increased apparent lipid and fatty acid digestibilities compared to RO or FO. These results demonstrate that the absorptive intestinal functions in fish can be altered by the feed oil history and that the effect remains after a return to a standard fish oil diet.     

Unexpected durability of PKH 26 staining on rat adipocytes

In a case of autologous mature fat cell transplantation to an individual rat, persistence of adipose staining with PKH26 was detected 14.5 months later. Fluorescent fat cells with spotty surface markings were easily visible. Such a long period of persistence and stability of staining exceeds the expectation from previous reports, and the given half-life of the substance. This finding encourages the use of the dye for long-term follow-up of connective tissue cells, especially adipocytes and preadipocytes, following transplantation.     

Species composition,abundance and emergence phenology of midges (Deiptera: Chironomidae) in a brown-water stream of West-Central Alberta,Canada

Sixteen floating, box type emergence traps, each covering 0.1 m², were placed along 150 m of a third-order stretch of the Bigoray River, a slow-flowing, vegetation-choked, brown-water muskeg stream. Effects of trap design, trap shading, length of sampling interval, and stream velocity on the number of midges caught was examined. Of the 112 species of Chironomidae caught, 32 species made up 90% of the catch and their emergence phenology is described in detail. There were more rare species than expected from Preston's lognormal distribution. Percentage of Bigoray species belonging to Tanypodinae, Orthocladiinae, Chironomini and Tanytarsini was 18%, 43%, 20%, and 19%, and was compared with 27 other studies on lotic chironomids. During the 140-day emergence period an average of 19.3 × 10³ chironomids emerged per square meter of stream. Based on changes in male:female ratios throughout a species emergence period, it was postulated that 30% of the Bigoray chironomid species were univoltine, 50% were bivoltine, and 20% were trivoltine.     

Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer

Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe ³H-labelled plasmid pABDI, which confers kanamycin resistance (Km^r) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Km^r gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks.