Influence of indoor microbial aerosol on the welfare of meat ducks.

The aim of the study was to evaluate the effects of microbial aerosols on ducks' welfare and provide information on which to establish microbial aerosol concentration standards for poultry. A total of 1800 1-d-old Cherry Valley ducks were randomly divided into 5 groups (A, B, C, D and E) with 360 ducks in each. To obtain objective data, each group had three replications. Different microbial aerosol concentrations in different groups were created by controlling ventilation and bedding cleaning frequency. Group A was the control group and hygienic conditions deteriorated progressively from group B to E. A 6-stage Andersen impactor was used to detect the aerosol concentration of aerobes, fungi, gram-negative bacteria and an AGI-30 microbial air sampler detected endotoxins. Physiological stress was evaluated in the ducks by adrenocorticotropic hormone (ACTH) values in serum. To assess the effects of bioaerosol factors, welfare indicators including fluctuating asymmetry (FA), appearance and gait as well as the Lactobacillus caecal concentration were evaluated. The data showed group D had already reached the highest limit of concentration of airborne aerobic bacteria, airborne fungi, airborne gram-negative bacteria and airborne endotoxin. The ducks in this group had significantly increased serum ACTH values and significantly decreased caecal lactobacilli concentration. Furthermore, appearance and gait scores, wing length and overall FA and caecal Lactobacillus concentration in this group were significantly increased at 6 and 8 weeks of age. In conclusion, high concentrations of microbial aerosol adversely affected the welfare of meat ducks. The microbial aerosol values in group D suggest a preliminary upper limit concentration of bioaerosols in ambient air for healthy meat ducks.

Seroprevalence of avian influenza H9N2 among poultry workers in Shandong Province, China.

H9N2 avian influenza virus has been circulating widely in birds, with occasional infection among humans. Poultry workers are considered to be at high risk of infection with avian influenza due to their frequent exposure to chickens, but the frequency of H9N2 avian influenza virus infections among them is still indistinct. This study was carried out in order to identify the seroprevalence of H9N2 avian influenza virus among poultry workers in Shandong, China. During the period from December 2011 to February 2012, a total of 482 subjects took part in this study, including 382 poultry workers and 100 healthy residents without occupational poultry exposure. Serum samples were collected and tested for the presence of antibodies against H9N2 avian influenza virus by hemagglutination inhibition (HI) and microneutralization (MN) assays. Nine subjects (9/382 = 2.3%) were positive for antibodies against H9N2 avian influenza virus among poultry workers by either HI or MN assays using ≥40 cut-off, while none of the 100 healthy residents were seropositive. In conclusion, our study identified H9N2 avian influenza infections among poultry workers in Shandong, China, and continuous surveillance of H9N2 avian influenza virus infection in humans should be carried out to evaluate the threat to public health.

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.

Occurrence of tetrodotoxin and paralytic shellfish poison in the Taiwanese crab Lophozozymus pictor.

Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in all 15 specimens of the xanthid crab Lophozozymus pictor collected from northern Taiwan in 1993. The average toxicity of crab specimens was 921 +/- 231 (mean +/- S.E.) mouse units. The toxin of crab was partially purified and then identified. It was found that the crab toxin contained TTX and gonyautoxin. The ratio of TTX to gonyautoxin for crab toxin was about 9:1.

Toxicity and toxic components of two xanthid crabs, Atergatis floridus and Demania reynaudi, in Taiwan.

Paralytic toxicity was detected by tetrodotoxin bioassay in eight specimens of Atergatis floridus and seven specimens of Demania reynaudi, collected from Taiwan in 1994. The toxicity of crab specimens was 161 +/- 115 (mean +/- S.D.) mouse units (MU) for A. floridus and 640 +/- 273 MU for D. reynaudi. The respective toxins were partially purified from specimens of A. floridus and D. reynaudi by ultrafiltration using a Diaflo YM-1 membrane, followed by chromatography on a Bio-Gel P-2 column. Electrophoresis, thin-layer chromatography, high-performance liquid chromatography, ultraviolet spectrum and gas chromatography-mass spectrometry indicated that the toxin of A. floridus was mainly composed of tetrodotoxin (85%), along with minor gonyautoxin 1-4 (15%), and the toxin of D. reynaudi was mainly composed of tetrodotoxin (88%), along with minor gonyautoxin 2-4 and neosaxitoxin (12%).

Occurrence of paralytic toxin in Taiwanese crab Atergatopsis germaini.

Paralytic toxicity was detected by paralytic shellfish poison bioassay for all 17 specimens of the xanthid crab A. germaini collected from northern Taiwan in November 1993. The average toxicity of crab specimens was 3809 +/- 2591 mouse units (mean +/- S.D.). The toxin was partially purified from ethanolic extract of the crab by ultrafiltration and Bio-Gel P-2 column chromatography. Electrophoresis, TLC, HPLC, ultraviolet spectrum and GC-MS analyses indicated that the crab toxin was composed of gonyautoxin 3 (50%), neosaxitoxin and saxitoxin (7%), a novel paralytic shellfish poison-like toxin (40%) and tetrodotoxin (3%).

ERIC-PCR identification of the spread of airborne Escherichia coli in pig houses.

To understand the spread of microbial aerosols in pig houses, with Escherichia coli (E. coli) as indicator, the airborne E. coli in 4 pig houses and their surroundings at different points 10, 50m upwind and 10, 50, 100, 200 and 400m downwind respectively from the pig houses were collected, and the concentrations were calculated at each sampling point. Furthermore, the feces of pigs were collected to separate E. coli. The ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction) technology was used to amplify the isolated E. coli DNA samples, then the amplified results were analyzed by NTSYS-pc (Version 2.10) to identify the similarity of isolated E. coli. The results showed that the airborne E. coli concentrations in indoor air of the 4 pig houses (21-35CFUm(-)(3) air) were much higher than those in upwind and downwind air (P<0.05), but there were no significant differences (P>0.05) at downwind distances. The ERIC-PCR results also showed that 52.4% of the fecal E. coli (four houses being respectively 2/4, 50%; 2/4, 50%; 3/6, 50%; 4/7, 57.1%) were identical to the indoor airborne E. coli isolates, and there was more than 90% similarity between the majority of E. coli (50%, 21/42) isolated from downwind air at 10, 50, 100 and 200m and those from indoor air or feces. It could be concluded that the aerosols in pig houses can spread to the surroundings, and thus effective measures should be taken to control and minimize the spread of microbial aerosols.

Usefulness of electrophoretic pattern of cell envelope protein as a taxonomic tool for fishhold slime moraxella species.

Nine independent Moraxella cultures were isolated from the accumulated slime in fishholds of fishery trawlers. It is significant that none of these isolates was viable above 30 degrees C, a temperature well below the usual incubation temperature for plate counts of food samples. The traditional taxonomic parameters showed no significant dissimilarities among these closely related marine organisms or between them and conventional moraxellas. However, cell envelope protein profiles examined on sodium dodecyl sulfate-polyacrylamide gels revealed that the organisms fell into several distinct groups. The cell envelope protein profile could be a simple and quick test to determine the fine relationships between individual isolates.

Characteristics of Escherichia coli grown in bay water as compared with rich medium.

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.

Occurrence of 2-keto-3-deoxy-D-manno-octonic acid in lipopolysaccharides isolated from Vibrio parahaemolyticus.

The occurrence of 2-keto-3-deoxy-D-manno-octonic acid (KDO) in lipopolysaccharides (LPS) of Vibrio parahaemolyticus was demonstrated for the first time by gas chromatography-mass spectrometry after dephosphorylation, reduction, and methylation. KDO was virtually completely phosphorylated, since no KDO was detected by either gas chromatography or thiobarbituric acid assay before dephosphorylation. The level of KDO in all six strains of V. parahaemolyticus investigated ranged from 0.37 to 0.69%, which was considerably lower than that in enterobacterial LPS.

Characteristics of heavily mucoid bacterial isolated from fish pen slime.

Weakly gram-positive pleomorphic rods isolated from the slime on the storage pen surfaces of fishing trawlers were found to produce extensive capsular material and intensely mucoid colonies. The isolates studied produced highly pleomorphic club-shaped cells indicative of coryneforms. Their DNA base composition ranged from 64.2 to 68.2 mol% guanine plus cytosine on the basis of melting point determinations.

Bactericidal activity of cerumen.

Freshly collected cerumen (dry form) suspended at a concentration of 3% in glycerol-sodium bicarbonate buffer showed bactericidal activity against some strains of bacteria tested. This suspension reduced the viability of Haemophilus influenzae, Escherichia coli K-12, and Serratia marcescens by more than 99%, whereas the viability of two Pseudomonas aeruginosa isolates, E. coli K-1, Streptococcus, and two Staphylococcus aureus isolates of human origin was reduced by 30 to 80%. The results support the hypothesis that cerumen functions to kill certain foreign organisms which enter the ear canal.

Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells.

Morphological changes of Vibrio parahaemolyticus from rods to spheres took place after a culture was subjected to starvation at a wide range of temperatures. Scanning electron micrographs revealed that starved spherical cells gradually developed a rippled cell surface with blebs and an extracellular filamentous substance adhesive to the cell surface. Cells starved at a low temperature for certain intervals were counted by various bacterial enumeration methods, including plate count, direct viable count, and total cell count for both Kanagawa-positive and -negative strains. The results indicated that this species could reach the nonculturable stage in 50 to approximately 80 days during starvation at 3.5 degrees C. Kanagawa-negative strain 38C6 lost culturability more slowly than Kanagawa-positive strain 38C1 at low temperature. As detected by thiosulfate-citrate-bile salts-sucrose plate count, a high percentage of the surviving cells at 3.5 degrees C in starvation medium were possibly injured by the low temperature rather than by starvation. Both addition of nalidixic acid to the starved cultures and the most-probable-number method demonstrated that the cells recovered after a temperature upshift probably represented the regrowth of a few surviving cells. These surviving cells were capable of growth and multiplication with limited nutrients at an extraordinary rate when the temperature was upshifted.

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus.

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.

Inactivation of bacteriophages by protein E, a new major membrane protein isolated from an Escherichia coli mutant.

Pure protein E, obtained after diethylaminoethyl-cellulose chromatography of ethylenediaminetetraacetic acid-Triton X-100-solubilized outer membrane proteins of Escherichia coli strain JF694, inactivated bacteriophage K3. Lipopolysaccharide enhanced bacteriophage inactivation. Antibody prepared against purified protein E protected bacteriophage K3 from inactivation by protein E. Bacteriophage K3 used a major outer membrane protein, protein II*, as part of its receptor. We conclude that proteins E and II* have a common region which interacts with bacteriophage K3. Protein E also inactivated two recently described bacteriophages, TC45 and TC23, that use protein E as at least part of their receptor.

Isolation and partial characterization of protein E, a major protein found in certain Escherichia coli K-12 mutant strains: relationship to other outer membrane proteins.

Escherichia coli outer membrane protein E was purified, and its amino acid composition and N-terminal amino acid were determined. The purified protein was shown to be immunologically and electrophoretically identical to proteins Ic (U. Henning, W. Schmidmayr, and I. Hindennach, Mol. Gen. Genet. 154:293-298, 1977) and e (W. van Alphen, N. van Selm, and B. Lugtenberg, Mol. Gen. Genet. 159:75-83, 1978). Proteins E, e, and Ic were also immunologically related to E. coli outer membrane protein Ia. Lugtenberg and co-workers (B. Lugtenberg, R. van Boxtel, C. Verhoef, and W. van Alphen, FEBS Lett. 96:99-105, 1978) have shown that electrophoretically identical peptides were generated by cyanogen bromide treatment of proteins E, e, and Ic.

New major outer membrane proteins found in an Escherichia coli tolF mutant resistant to bacteriophage TuIb.

Cell envelopes prepared from an Escherichia coli tolF strain selected as resistant to phage TuIb contained a new major outer membrane protein related to outer membrane proteins Ia and Ib. The strain that produces this protein is a tolF par double mutant but contains an additional mutation leading to the production of the new major outer membrane protein. Antibiotic sensitivity lost as a result of the tolF mutation is regained in strains that contain the new major outer membrane protein. This indicates that this protein functions to restore the selective permeability of the outer membrane to low-molecular-weight hydrophilic molecules.

Chromosomal location of a gene (nmpA) involved in expression of a major outer membrane protein in Escherichia coli.

The phenotypic expression of protein E, a recently described major outer membrane protein, is associated with a mutation at a locus on the Escherichia coli chromosome that we call nmpA. nmpA is located between rbsK and uncA at 82.7 min on the E. coli linkage map. The nmpA locus is also the site of the mutations which lead to the formation of major outer membrane proteins Ic or e. It is likely proteins E, Ic, and e are closely related or identical. The mutant nmpA allele is dominant.

Defeat of colicin tolerance in Escherichia coli ompA mutants: evidence for interaction between colicin L-JF246 and the cytoplasmic membrane.

Escherichia coli ompA mutants are tolerant to colicin L-JF246. This tolerance can be overcome by a variety of treatments that have as their target the outer membrane or the peptidoglycan layers of the cell envelope. Thus, increasing the concentration of colicin L, releasing lipopolysaccharide from the outer membrane by treatment of intact cells with ethylenediaminetetracetic acid (EDTA), converting cells to spheroplasts by treatment with lysozyme-EDTA or penicillin, or trypsin, treatment of intact cells will result in an increased colicin sensitivity. These treatments alter the outer membrane of ompA mutants and suggest that the altered outer membrane may allow the penetration of at least a portion of the colicin L molecule to a site of action located within this barrier. To substantiate this, we have demonstrated that membrane vesicles prepared from ompA mutants are sensitive to colicin L and that 14C-labeled colicin L binds rapidly to both the outer and inner membrane fractions of the cell.