Expression and characterization of a recombinant i-type lysozyme from the harlequin ladybird beetle Harmonia axyridis.

Lysozymes are enzymes that destroy bacterial cell walls by hydrolysing the polysaccharide component of peptidoglycan. In insects, there are two classes of lysozymes, the c-type with muramidase activity and the i-type whose prototypical members from annelids and molluscs possess both muramidase and isopeptidase activities. Many insect genes encoding c-type and i-type lysozymes have been identified during genome and transcriptome analyses, but only c-type lysozymes have been functionally characterized at the protein level. Here we produced one of five i-type lysozymes represented in the immunity-related transcriptome of the invasive harlequin ladybird beetle Harmonia axyridis as recombinant protein. This was the only one containing the serine and histidine residues that are thought to be required for isopeptidase activity. This i-type lysozyme was recombinantly expressed in the yeast Pichia pastoris, but the purified protein was inactive in both muramidase and isopeptidase assays. Transcription and immunofluorescence analysis revealed that this i-type lysozyme is produced in the fat body but is not inducible by immune challenge. These data suggest that i-type lysozymes in insects may have acquired novel and as yet undetermined functions in the course of evolution.

Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria.

To eradicate rabies in foxes, almost 97 million oral rabies vaccine baits have been distributed in Germany and Austria since 1983 and 1986, respectively. Since 2007, no terrestrial cases have been reported in either country. The most widely used oral rabies vaccine viruses in these countries were SAD (Street Alabama Dufferin) strains, e.g. SAD B19 (53.2%) and SAD P5/88 (44.5%). In this paper, we describe six possible vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) detected during post-vaccination surveillance from 2001 to 2006, involving two different vaccines and different batches. Compared to prototypic vaccine strains, full-genome sequencing revealed between 1 and 5 single nucleotide alterations in the L gene in 5 of 6 SAD isolates, resulting in up to two amino acid substitutions. However, experimental infection of juvenile foxes showed that those mutations had no influence on pathogenicity. The cases described here, coming from geographically widely separated regions, do not represent a spatial cluster. More importantly, enhanced surveillance showed that the vaccine viruses involved did not become established in the red fox population. It seems that the number of reported vaccine virus-associated rabies cases is determined predominantly by the intensity of surveillance after the oral rabies vaccination campaign and not by the selection of strains.

Characterization of a repetitive DNA fragment in Hammondia hammondi and its utility for the specific differentiation of H. hammondi from Toxoplasma gondii by PCR.

Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites. Both species use felids as definitive hosts and a broad spectrum of warm-blooded animals as intermediate hosts. Morphologically and serologically, the two parasites are difficult to differentiate. While T. gondii is an important pathogen of humans and a broad range of other vertebrates, disease has not yet been associated with H. hammondi infection. The aim of the present study was to identify and characterize a repetitive DNA fragment in H. hammondi and to evaluate its suitability for diagnostic purposes. With two primers considered to be specific for a 529 bp repetitive DNA fragment in T. gondii, weak products were amplified by polymerase chain reaction (PCR) from genomic DNA from H. hammondi oocysts. These amplicons (of approximately 150, 300 and 450 bp) were sequenced. The 292 bp consensus sequence of these three fragments revealed 84% identity with parts of the 529-bp repeat in T. gondii. Based on this sequence, a pair of primers was selected which amplified products of 98 and 630 bp from genomic DNA from H. hammondi oocysts but not from DNA from T. gondii. The 630-bp product was purified and cloned into a plasmid vector and the consensus sequence determined from seven randomly selected clones; comparison of this sequence with those available in current databases for T. gondii revealed an 84.0-88.1% identity over a length of 529 bp. The sequence data obtained was used for the development of a sensitive PCR which is entirely specific for H. hammondi and incorporates an internal control. The sequence data for the repetitive DNA element of H. hammondi provides a foundation for the design of primers specific to T. gondii, and the future optimisation of conventional and real-time PCR assays for the specific diagnosis of toxoplasmosis in definitive and intermediate hosts.

Combination effect of ceftriaxone with four aminoglycosides on nonfermenting gram-negative bacteria.

The in vitro efficacy of ceftriaxone in combination with gentamicin, tobramycin, amikacin and netilmicin against 50 nonfermenting gram-negative bacterial strains was compared by use of the checkerboard agar dilution technique. On average 42.5% of all nonfermenting strains were inhibited by additive, 22.5% by synergistic ceftriaxone-aminoglycoside combinations. Great variations occurred between the different bacterial species. Ceftriaxone-tobramycin interactions were superior to combinations with other aminoglycosides. Ceftriaxone-aminoglycoside combinations were most active on Pseudomonas aeruginosa and least potent on Pseudomonas cepacia.

Circulating diiodotyrosine: studies of its serum concentration, source, and turnover using radioimmunoassay after immunoextraction.

This report describes the application of a sensitive, specific, and reproducible RIA for diiodotyrosine (DIT) in human serum and metabolic studies on the source and kinetics of circulating DIT. Interference by cross-reactivity of T4 and other analogs was completely eliminated by isolation of DIT from serum with an efficient preparative immunoprecipitation technique. Mean (+/- SD) serum DIT levels were 161 +/- 133 pmol/liter (7.0 ng/100 ml) in 41 normal subjects, 64 +/- 30 pmol/liter in 46 pregnant women, 241 +/- 83 pmol/liter in the cord serum of 48 newborn infants, 542 +/- 494 pmol/liter in 22 hyperthyroid patients, and 101 +/- 71 pmol/liter in 15 hypothyroid patients. Mean values in pregnant, newborn and hyperthyroid subjects were significantly different from the normal mean. Very low DIT serum levels were found in four athyreotic patients during oral T4 substitution therapy, indicating that little DIT is formed by peripheral T4 degradation. In five normal subjects who received a single oral dose of 3 mg T4, serum DIT remained unchanged in one case and decreased in four cases. Radioimmunological measurements of DIT elimination from serum after the iv injection of 1 mg DIT in two normal volunteers gave MCRs of 103 and 133 liters/day and an average extrathyroidal DIT turnover rate of 19 nmol/day (8.2 microgram/day). These data indicate that circulating DIT arises predominantly from the thyroid, suggesting that peripheral formation of DIT is a minor metabolic pathway in the human.

Comparison of recommended respirable mass dust sampling devices.

The draft, "Revised Guide for Respirable Mass Sampling," prepared by the AIHA-ACGIH Aerosol Hazards Committee, recommends five respirable mass sampling devices that sample in accordance with the "Los Alamos" respirable dust criterion. The spectrum of recommended sampling rates for these devices ranges from 1.8 to 430. liters per minute. A study was conducted to determine if simultaneous measurements with these devices yielded comparable respirable mass concentrations. The penetration characteristics of the first stage separators were also evaluated and compared.

Quantitating the efficiency of the coal mine personal respirable dust sampler for sampling oxide fumes.

Coal mine environments containing fume contaminants are assessed using the standard personal respirable dust sampler. The effect of the first-stage collector and filter on the total concentration measured was evaluated. The results showed that the personal respirable dust sampler could be used to sample fumes with TLV's greater than 1.0 mg/m3.