Global and Phylogenetic Distribution of Quorum Sensing Signals,Acyl Homoserine Lactones,in the Family of Vibrionaceae

Bacterial quorum sensing (QS) and the corresponding signals, acyl homoserine lactones (AHLs), were first described for a luminescent Vibrio species. Since then, detailed knowledge has been gained on the functional level of QS; however, the abundance of AHLs in the family of Vibrionaceae in the environment has remained unclear. Three hundred and one Vibrionaceae strains were collected on a global research cruise and the prevalence and profile of AHL signals in this global collection were determined. AHLs were detected in 32 of the 301 strains using Agrobacterium tumefaciens and Chromobacterium violaceum reporter strains. Ethyl acetate extracts of the cultures were analysed by ultra-high performance liquid chromatography-high resolution mass spectrometry (MS) with automated tandem MS confirmation for AHLs. N-(3-hydroxy-hexanoyl) (OH-C6) and N-(3-hydroxy-decanoyl) (OH-C10) homoserine lactones were the most common AHLs found in 17 and 12 strains, respectively. Several strains produced a diversity of different AHLs, including N-heptanoyl (C7) HL. AHL-producing Vibrionaceae were found in polar, temperate and tropical waters. The AHL profiles correlated with strain phylogeny based on gene sequence homology, however not with geographical location. In conclusion, a wide range of AHL signals are produced by a number of clades in the Vibrionaceae family and these results will allow future investigations of inter- and intra-species interactions within this cosmopolitan family of marine bacteria.     

Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

Concentrations of trimethoprim and sulphadoxine in tissues from goats and a cow.

The concentration of trimethoprim and sulphadoxine in plasma and tissue from goats and a cow have been determined after a single intravenous injection. Furthermore, the concentration of the two drugs and their metabolites in plasma and tissues have been determined after continuous intravenous infusion for 2½–3 hrs. Trimethoprim was present in all tissues but brain at higher concentrations than in plasma while the concentration of sulphadoxine in the different tissues were lower than in plasma. The highest concentration of the 2 drugs and their metabolites was found in the kidney. The distribution pattern of trimethoprim and sulphadoxine was similar in cow and goats.     

Detection of tobacco rattle virus in potato tubers using a simple RT-PCR procedure

Summary A simple assay for detection of tobacco rattle virus in infected tubers involving RT-PCR was developed. The assay detected a wide range of strains, including nonparticle NM variants. RNA extraction methods were compared, and a simple method was found to give reliable results. The method enabled detection of at least 1 ng of tobacco rattle particles in 100 mg tuber extract.     

Screening for infection of Trichinella in red fox (Vulpes vulpes) in Denmark

A total of 6141 foxes (Vulpes vulpes) were examined for infection with Trichinella. The foxes were killed in Denmark during the hunting season 1995-1996 and 1997-1998; 3133 and 3008, respectively. Foxes included in the investigation came from throughout the country with the exception of the island of Bornholm. The right foreleg from each fox was submitted for investigation. The legs were stored at -20 degrees C for 3-10 months prior to examination. Following thawing, muscle tissue (10 g) from each leg was examined by trichinoscopy and by a pepsin-HCl digestion technique. In 1995-1996, three foxes were found positive corresponding to a prevalence of 0.001. Each of the infected foxes harboured an extremely low infection, i.e. about one larva per 10 g muscle tissue. It was not possible to obtain sufficient larval material for species identification. All three foxes were shot in the vicinity of a small village in the north-western part of Denmark. In 1997-1998 no Trichinella cases were found. The results, compared with previous studies, indicate that the prevalence of infection of Trichinella sp. among wild living foxes in Denmark is very low. This is further supported by the fact, that no infection of Trichinella sp. has been found in slaughtered pigs in Denmark for more than 65 years, which suggests that the infection pressure is very low. Considering the facts above we conclude that the risk of Trichinella infections is negligible in intensive indoor pig production units in Denmark whereas high local prevalence of Trichinella infections in the wildlife might constitute a serious risk for the expanding outdoor pig production.     

Respiratory tract distribution and bioavailability of spiramycin in calves

Pharmacokinetic determinants of spiramycin and its distribution into the respiratory tract were studied in 2 groups of calves, 4 to 10 weeks old. Group-A calves (n = 4) were used to determine pharmacokinetic variables of spiramycin after IV (15 and 30 mg/kg of body weight) and oral administrations of the drug (30 mg/kg) and to measure distribution of spiramycin into nasal and bronchial secretions. Group-B calves (n = 4) were used to determine distribution of spiramycin into lung tissue and bronchial mucosa. Spiramycin disposition was best described by use of an open 3-compartment model. Mean (+/- SD) elimination half-life was 28.7 +/- 12.3 hours, and steady-state volume of distribution was 23.5 +/- 6.0 L/kg. Bio-availability after oral administration was 4 +/- 3%. High and persistent concentrations of spiramycin were achieved in the respiratory tract tissues and fluids. Tissue-to-plasma concentration ratio was 58 for lung tissue and 18 for bronchial mucosa at 3 hours after spiramycin administration and 137 and 49, respectively at 24 hours. Secretion-to-plasma concentration ratio was 4 for nasal secretions and 7 for bronchial secretions, and remained almost constant with time. Thus, spiramycin penetrates well into the respiratory tract, although the value in bronchial secretions is lower than that in lung tissues and bronchial mucosa. Calculations indicate that a loading dose of 45 mg/kg, administered IV, followed by a maintenance dose of 20 mg/kg, IV, once daily is required to maintain active concentrations of spiramycin against bovine pathogens in bronchial secretions.     

No transmission of Potato spindle tuber viroid shown in experiments with thrips (Frankliniella occidentalis, Thrips tabaci), honey bees (Apis mellifera) and bumblebees (Bombus terrestris)

Experiments were carried out to investigate whether Potato spindle tuber viroid (PSTVd) can be transmitted intra- and inter-species from infected Solanum jasminoides to non-infected S. jasminoides and S. esculentum and from infected Brugmansia sp. to S. esculentum by Frankliniella occidentalis and Thrips tabaci by leaf sucking. The F. occidentalis experiments also included feeding on pollen prior to feeding on PSTVd-infected leaf. No thrips-mediated transmission of PSTVd was recorded. The possibility of PSTVd transmission by Apis mellifera and Bombus terrestris during their feeding/pollinating activities within ornamentals and from ornamentals to S. esculentum was included, and no bee-mediated transmission was recorded.     

Chlorophyll inheritance patterns and extent of natural self-pollination in timothy

Three hundred sixty-five S₁ and 234 S₂ seedling populations of timothy (Phleum pratense L.) were examined for their segregations for chlorophyll deficiencies. Arrays of both self-and open-pollination families indicated a lack of concentration at definite levels, with ratios expressed forming a continuous series from 4.6:1 to 572:1 for green: chlorophyll-deficient types in the S₁ generation. The S₂ populations examined ranged from 1.8:1 to 244:1. Arrays were very similar in their distributions.Sixty-three plants yielded chlorophyll-deficient types in both S₁ and S₀ populations. From these, it was calculated that 7 to 8 percent of natural self-pollination may occur in timothy.Except at two levels (28:1 and 143:1 tetradisomic) assumption of tetradisomic or simpler inheritance patterns resulted in lower calculated percentages of natural self-pollination than resulted from assumed hexasomic inheritance. No apparent explanation is available to account for these divergences from the regular pattern encountered.Results of cooperative work of the Crops Research Division, Agriculture Research Service, U.S. Department of Agriculture and the Wisconsin Agricultural Experiment Station, Madison.Research Agronomist, Crops Research Division, Agriculture Research Service, U.S. Department of Agriculture and Associate Professor, and Professor of Agronomy, Wisconsin Agricultural Experiment Station, respectively.