Biomedical subjects

L B Selinger

Publications and source records attributed to L B Selinger.

At least 19 recordsLinked to original sources

PubMed2005

Retention of Escherichia coli by house fly and stable fly (Diptera: Muscidae) during pupal metamorphosis and eclosion.

Populations of Escherichia coli obtained by feeding larval house flies, Musca domestica L. and stable flies, Stomoxys calcitrans (L.), persisted through the pupal stage. The abundance of E. coli in house fly pupae increased initially then declined before adult emergence. Abundance of E. coli in stable fly pupae increased through pupal development and remained high. Infected stable fly pupal cases typically contained more E. coli than house fly pupal cases. A greater proportion of emerging adult house flies were infected with E. coli compared with stable flies; however, the abundance of E. coli on infected flies was similar between species. Adult flies contained 0.04-0.19% of the E. coli in the pupal cases. The proportion of infected house fly adults and the amount of E. coli on the infected flies were related to the levels of E. coli in the pupal cases; however, these relationships did not occur with the stable fly. Results suggest that retention of E. coli from larval to adult house flies could play a role in the transmission and spread of E. coli, whereas stable fly adults probably play a minor role in E. coli spread. However, pupae of both species have potential to act as reservoirs for E. coli.

Animals↗

PubMed2004

Persistence of Escherichia coli in immature house fly and stable fly (Diptera: Muscidae) in relation to larval growth and survival.

The persistence of Escherichia coli in artificially fed larvae was examined for up to 48 h after ingestion by house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). The rate of change in the E. coli load was similar for both species for up to 5 h after ingestion. Up to 48 h after ingestion, abundance of E. coli declined in immature house flies but remained constant in immature stable flies. When different E. coli concentrations were fed to larvae, the abundance of E. coli increased in stable fly larvae regardless of the initial concentration. The E. coli load in house fly larvae increased when larvae were fed a low concentration of bacteria, but it declined when larvae were fed a high concentration of bacteria. Survival of house fly and stable fly larvae averaged 62 and 25%, respectively, when reared on pure E. coli cultures. These observations suggest that house fly larvae digest E. coli and use it as a food source but stable fly larvae do not.

Alberta↗

PubMed2003

Transfer of a rifampicin-resistant Escherichia coli strain among feedlot cattle.

AIMS: To determine the incidence of transfer of a naturally occurring rifampicin-resistant strain of Escherichia coli (RREC) among cattle in a research feedlot. METHODS AND RESULTS: During three separate experiments, steers in three different pens were orally inoculated with RREC originally isolated from bovine faeces. Faecal swabs were performed on all steers in the feedlot at approximately 5 week intervals thereafter. Faecal grab samples were collected from steers in the inoculated and the immediately adjacent pens for up to 4 months. In all three experiments, the inoculated steers and penmates shed RREC within 48 h, and then shed intermittently throughout the sampling periods. Transfer of RREC to steers in an adjacent pen was confirmed only during the first experiment, but never to those in non-adjacent pens. All recovered RREC isolates were compared with the inoculated strain using multiple methods indicating that all RREC isolates were descendants of the original inoculated strain. CONCLUSIONS: Detection of the RREC strain on the pen floor and within the animal handling system, but not in the feed troughs or water bowls, suggests faecal-oral to be the primary mode of transmission among animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that in the absence of selective pressure, antibiotic-resistant bacteria may persist in cattle for a short duration but widespread transfer among cattle in a feedlot environment may be an exception rather than the norm. Modifications to feedlot management are discussed.

Animal Feed↗

PubMed2003

Prevalence of Escherichia coli O157:H7 and Salmonella spp. in surface waters of southern Alberta and its relation to manure sources.

The Oldman River watershed in southern Alberta, Canada, is an extensively irrigated region in which intensive agricultural practices have flourished. Concern over water quality in the basin has been expressed because of high levels of enteric disease indigenous to the region. To address these concerns, we conducted a 2-year study to estimate the prevalence of Escherichia coli O157:H7 and Salmonella spp. in surface water within the basin. This study is the first of its kind to identify E. coli O157:H7 repeatedly in surface water collected from a Canadian watershed. Prevalence of E. coli O157:H7 and Salmonella spp. in water samples was 0.9% (n = 1,483) and 6.2% (n = 1,429), respectively. While data examined at a regional level show a relationship between high livestock density and high pathogen levels in southern Alberta, statistical analysis of point source data indicates that predicted manure output from bovine, swine, and poultry feeding operations was not directly associated with either Salmonella spp. or E. coli O157:H7 prevalence. However, geography and weather variables, which are likely to influence bacterial runoff, were not considered in this model. We also postulate that variations in time, amount, and frequency of manure application onto agricultural lands may have influenced levels of surface-water contamination with these bacterial pathogens.

Alberta↗

PubMed2002

Evidence of quorum sensing in the rumen ecosystem: detection of N-acyl homoserine lactone autoinducers in ruminal contents.

Acyl-homoserine lactone (AHL) based quorum-sensing systems are widespread among gram-negative bacteria, particularly in association with plants and animals. As yet, there have been no reports of AHL signaling in the anaerobic rumen environment, an ecosystem of great complexity in which cell-cell signaling is likely to occur. We detected multiple AHL autoinducers in the rumen contents of 6 out of 8 cattle fed a representative selection of diets. The signals were not associated with feed. Surprisingly, no pure cultures produced AHLs in vitro when grown under the laboratory conditions we tested. Our observations suggest that either (a) a factor specific to the rumen ecosystem is required for the rumen isolates we tested to produce AHLs or (b) a strain (or strains) that we were not able to culture but which grows to a high cell density in the rumen produces the AHLs we detected.

4-Butyrolactone↗

PubMed2002

Comparsion of selected growth media for culturing Serratia marcescens, Aeromonas sp., and Pseudomonas aeruginosa as pathogens of adult Stomoxys calcitrans (Diptera: Muscidae).

Stable flies, Stomoxys calcitrans (L.), were orally infected with Aeromonas sp., Pseudomonas aeruginosa (Schroeter), and Serratia marcescens Bizio that were cultured on egg-yolk media, nutrient broth, and fly egg media. Aeromonas and Serratia caused mortality when the bacteria were originally grown on egg-yolk medium. Pseudomonas was equally lethal regardless of the media on which it was cultured. A wild isolate of Aeromonas caused greater death than an isolate that had been passed through host flies and had been reisolated from killed flies. Mortality increased with bacterial dose for all species. Aeromonas and Serratia caused mortality within several days after ingestion, whereas Pseudomonas caused a gradual increase in mortality 3-7 d after ingestion. The pathologic activity of Aeromonas and Serratia required extracellular products produced when cells were grown in egg yolk medium. Aeromonas required both supernatant and cells from egg yolk medium, wereas Serratia required supernatant from egg yolk medium and cells from either nutrient broth or egg yolk medium. Mortality due to ingestion of Aeromonas was correlated with the presence of enzymes that cause alpha- and beta-hemolysis, while mortality following ingestion of Serratia was associated with alpha-hemolysins, elastases, and chitinases.

Aeromonas↗

PubMed2001

Growth and survival of immature Heamatobia irritans (Diptera; Muscidae) is influenced by bacterial isolated from cattle manure and conspecific larvae.

Twenty species of bacteria were isolated from cattle manure and seven species were isolated from the gut of larval horn fly Hematobia irritans (L.). Bacteria in manure belonged to the Bacillaceae, Pseudomonadaceae, Micrococcaceae, Corynebacteriaceae, Enterobacteriaceae, Microbacteriaceae, and two unassigned genera. Gut bacteria belonged to the Enterobacteriaceae, Bacillaceae, Neisseriaceae, and Pseudomonadaceae. H. irritans larval survival and growth on the various bacterial species were evaluated by rearing larvae in sterilized cattle manure that was inoculated with single bacterial isolates. H. irritans larvae failed to develop in sterilized, uninoculated manure, indicating that bacteria are necessary for larval development. Survival averaged 74% in nonsterilized manure and ranged from 4 to 53% in manure with individual isolates. Survival was highest when larvae were reared on manure inoculated with Pseudomonadaceae, Corynebacteriaceae, Micrococcaceae, and Bacillaceae and was lowest when reared in manure inoculated with Enterobacteriaceae and Microbacteriaceae. Pupal weights were heaviest when reared on the Flavobacteria, followed by the Pseudomonadaceae and Corynebacteriaceae. Pupae averaged 4.9 +/- 0.08 mg when reared on gram-negative isolates, compared with 3.6 +/- 0.09 mg when reared on gram-positive isolates. Pupal weights were not significantly correlated with larval survival, indicating that bacteria that promote growth do not necessarily promote survival. A reproductive index was used as a measure of fitness and was highest for larvae reared in the nonsterile control, followed most closely by Pseudomonadaceae and Corynebacteriaceae. These groups appeared to best meet the nutritional requirements of larvae and may be used in further experiments to define an artificial rearing media for H. irritans.

Actinomycetales↗

PubMed2001

An improved method to illuminate and record a phytase assay in SDS-PAGE zymogram gels.

Experimentation at the Lethbridge Research Center in Alberta, Canada using cross-polarized transmitted light to photographically record a staining technique on zymograms has proved to be successful with both color and black and white films. It has been possible to obtain the desired visible contrast without compromising the intensity of the enzyme activity bands. Increasing numbers of such PAGE gels are being submitted for photographic recording when it is believed that the image will be used for records, publication, scientific posters or AV presentations.

6-Phytase↗

PubMed2000

Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy.

The localization of phytase (myo-inositol-hexaphosphate phosphohydrolase) in the ruminal bacteria, Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2), was determined with transmission electron microscopy. Phosphate produced from the enzymatic dephosphorylation of the calcium salt of phytic acid is precipitated as calcium phosphate. The calcium is then replaced with lead to produce electron-dense lead phosphate. This deposition of lead phosphate localized phytase in S. ruminantium JY35 and M. multiacidus 46/5(2) to the outer membrane, and confirmed intracellular expression of the enzyme in Escherichia coli pSrP.2, the recombinant clone which possesses the gene (phyA) encoding phytase (phyA) in S. ruminantium.

6-Phytase↗

PubMed2000

Production of cellulases and xylanases by low-temperature basidiomycetes.

Three of four isolates, representing phylogenetically distinct groupings of low-temperature basidiomycetes (LTB), were capable of utilizing wheat straw, and to a lesser extent conifer wood at 15 degrees C. A cottony snow mould LTB (LRS 013) and a fruit rot LTB (LRS 241) grown on straw significantly degraded filter paper, carboxymethylcellulose (CMC), p-nitrophenyl beta-glucopyranoside (i.e., beta-glucosidases), and xylan. Enzymes produced by Coprinus psychromorbidus (LRS 067) were limited to xylanases from straw and wood and beta-glucosidases from wood. A sclerotia-forming LTB (LRS 131) exhibited poor growth on both substrates, and did not produce detectable quantities of extracellular enzymes. None of the LTB isolates tested degraded avicel. The temperature optima of CMCases and xylanases in the filtrates from the straw medium ranged from 25 degrees C to 55 degrees C, and with the exception of LRS 067, significant activity was observed at 5 degrees C. Two cellulases (25 and 31 kDa) and two xylanases (24 and 34 kDa) were observed on zymograms for LRS 013 and 241. Reduction of enzymes with 2-mercaptoethanol adversely affected their activity on zymograms, and an additional cellulase band was observed for non-reduced samples. This study indicates that LTB produce an array of cellulolytic and xylanolytic enzymes, and that some of these enzymes possess low-temperature optima which may facilitate degradation of plant fibre under low-temperature conditions.

Basidiomycota↗

PubMed1999

A novel staining method for detecting phytase activity.

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

6-Phytase↗

PubMed1999

Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium.

The growth and survival of Stomoxys calcitrans (L.) larvae on egg yolk medium inoculated with bacteria isolated from a colony of stable flies was evaluated. Five species of bacteria--Acinetobacter sp., Aeromonas sp., Empedobacter breve (Holmes & Owen), Flavobacterium odoratum Stutzer, and Serratia marcescens Bizio--were identified according to fatty acid profiles using a microbial identification system. Larvae failed to develop on uninoculated plates, confirming that bacteria are required to complete development. Larvae also failed to complete development on plates inoculated with Aeromonas sp. and S. marcescens, and died during the 1st instar. Larvae completed development on the remaining 3 bacterial species as well as on Escherichia coli (Migula). Survival was generally higher when larvae were reared on Acinetobacter sp. and F. odoratum compared with E. coli and E. breve. Egg density did not influence larval survival, although the variability in survival was lowest using 20 and 40 eggs per plate. Larval survival in mixed cultures of Acinetobacter and Flavobacterium averaged 22.7% lower than survival in the pure cultures, and averaged 21.6% higher in mixed cultures of Empedobacter and Flavobacterium compared with pure cultures. Larval survival in mixed cultures did not differ significantly from mean survival in pure cultures for combinations of Acinetobacter and E. coli, Acinetobacter and Empedobacter, E. coli and Empedobacter, and E. coli and Flavobacterium. Larval developmental time was faster on all mixed bacterial cultures compared with developmental time on pure bacterial cultures. Optimal sample sizes and egg numbers are presented for detecting specified differences in larval survival. This rearing procedure will be useful for studying insect-microbe interactions and evaluating mortality using bacterial agents.

Animals↗

PubMed1998

Phytase activity of anaerobic ruminal bacteria.

Phytase catalyses the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. The presence of phytase activity was investigated in 334 strains of 22 species of obligately anaerobic ruminal bacteria. Measurable activities were demonstrated in strains of Selenomonas ruminantium, Megasphaera elsdenii, Prevotella ruminicola, Mitsuokella multiacidus and Treponema spp. Strains isolated from fermentations with cereal grains proved to have high activity, and activity was particularly prevalent in S. ruminantium, with over 96% of the tested strains being positive. The measured phytase activity was found exclusively associated with the bacterial cells and was produced in the presence of approximately 14 mM phosphate. The most highly active strains were all S. ruminantium, with the exception of the one Mitsuokella multiacidus strain examined. Phytase activity varied greatly among positive strains but activities as high as 703 nmol phosphate released (ml culture)-1 were measured for a S. ruminantium strain and 387 nmol phosphate released (ml culture)-1 for the Mitsuokella multiacidus strain.

6-Phytase↗

PubMed1998

Expression of a Bacillus thuringiensis delta-endotoxin gene by Bacillus pumilus.

The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delta-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and delta-endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that < 0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of delta-endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level delta-endotoxin production occurred concomitant with sporulation. Synthesis of functional delta-endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of delta-endotoxins to the root environment for biocontrol purposes.

Animals↗

PubMed1997

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM.

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.

Amino Acid Sequence↗

PubMed1997

Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39.

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.

Amino Acid Sequence↗

PubMed1997

An endoglucanase from the anaerobic fungus Orpinomyces joyonii: characterization of the gene and its product.

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 degrees C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.

Amino Acid Sequence↗

PubMed1996

The rumen: a unique source of enzymes for enhancing livestock production.

Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed at increasing production efficiency. One particularly promising technology is feeding enzymes as supplements for animal diets. Supplementation of diets for non-ruminants (e.g., swine and poultry) with fibrolytic enzymes, such as cellulases, xylanases and beta-glucanases, increases the feed conversion efficiency and growth rate of the animals. Enzymatic hydrolysis of plant cell wall polymers (e.g., cellulose, xylan, beta-glucans) releases glucose and xylose and eliminates the antinutritional effects of beta-glucans and arabinoxylans. Enzyme supplementation of diets for ruminants has also been shown to improve growth performance, even though the rumen itself represents the most potent fibrolytic fermentation system known. Implementation of this technology in the livestock industry has been limited largely because of the cost of development and production of enzymes. Over the last decade, however, developments in recombinant DNA technology have increased the efficiency of existing microbial production systems and facilitated exploitation of alternative sources of industrial enzymes. The ruminal ecosystem is among the novel enzyme sources currently being explored. Understanding the role of enzymes in feed digestion through characterization of the enzymology and genetics involved in digestion of feedstuffs by ruminants will provide insight required to improve the products currently available to producers. Characterization of genes encoding a variety of hydrolytic enzymes, such as cellulases, xylanases, beta-glucanases, amylases, pectinases, proteases, phytases and tannases, will foster the development of more efficacious enzyme supplements and enzyme expression systems for enhancing nutrient utilization by domestic animals. Characteristics of the original source organism need no longer restrict the production of a useful enzyme. Recent reports of transgenic plants expressing fibrolytic or phytase activity and of transgenic mice able to produce endoglucanase in the pancreas speak to the feasibility of improving feed digestion through genetic modification of the feedstuffs and the animals.

Journal Article↗