Precision and accuracy of a point-of-care glucometer in horses and the effects of sample type.

Point-of-care glucometry is used commonly in clinical and research settings; however, accuracy and precision of this method are concerns. The objectives of this study were to determine the accuracy of glucometry in adult horses and the precision of duplicate measurements. Blood samples were collected from 62 horses into one plain syringe, one EDTA tube and three fluoride oxalate (FO) tubes. Immediately after collection, glucose concentrations in whole blood were determined, in duplicate, by glucometry from the syringe (plain whole blood [WB] group), EDTA tube (EDTA group) and one FO tube (FO group). One FO sample was used to measure plasma glucose concentration by a laboratory chemistry analyser (LAB group) ≤1 h after collection. The third FO tube was used to measure plasma glucose concentration by glucometry after 3 h storage (FO3hr group). Adequate precision was present for all groups (coefficient of variation: 0.7-3.5%) except WB (5.5-9.4%). Between groups, correlations were significant (P < 0.05; except for WB-EDTA), varied with group comparison, and tended to be lowest for comparisons involving WB. Mean bias was lowest for WB-LAB and greatest for FO-LAB and FO3hr-LAB; however, the limits of agreement were ≥4.65 mmol/L for WB-LAB and ≤2.75 mmol/L for most other comparisons. For the glucometer used, performance was influenced by sample type: WB was unsuitable, while FO or EDTA samples resulted in adequate precision and accuracy, provided under-estimation of glucose concentrations is accounted for by using method-specific reference ranges. Glucometer performance and optimal sample type(s) should be determined prior to use in horses.

The efficacy of ivermectin, pyrantel and fenbendazole against Parascaris equorum infection in foals on farms in Australia.

This study was performed to estimate the prevalence of patent Parascaris equorum infections and determine the efficacy of ivermectin, pyrantel and fenbendazole against P. equorum infection in foals on farms in southern Australia. Foals aged >3 months on five farms in the south-western slopes region of New South Wales were used. Faeces were collected from each foal and foals with a P. equorum faecal egg count (FEC) of >100 eggs per gram (EPG) were used to measure anthelmintic efficacy using the FEC reduction (FECR) test, after random allocation to a control group or an ivermectin, pyrantel embonate or fenbendazole treatment group. Treatment was administered on day 0 and faeces were collected on day 14 and a FEC was performed. For determination of anthelmintic efficacy, FECRs and lower 95% confidence intervals (LCL) were calculated using previously described methods, based on individual or group FECRs. P. equorum populations were considered susceptible when FECR was >90% and LCL >90%, suspected resistant when FECR was FECR was 80-90% and LCL <90% and resistant when FECR was <80% and LCL <90%. A Poisson distribution quality control method was applied to the data to remove suspected erroneous FECR results. Prevalence of patent P. equorum infection was 58.3% (147/252 foals) and 89 foals on 5 farms were included in the FECR study. Resistance of P. equorum to ≥ 1 anthelmintic was present on all five farms prior to and on four farms after application of the quality control method. Two farms had evidence of multiple drug resistance. Ivermectin was effective and ineffective on two and three farms, respectively. Fenbendazole was effective on two farms, equivocal on one farm and ineffective on one farm. Pyrantel embonate was effective on three farms and ineffective on one farm. These data indicate that anthelmintic-resistant P. equorum populations are present on farms in Australia and multiple drug resistance may occur on individual farms.

Fibrinous pericarditis and pericardial effusion in three neonatal foals.

CASE SERIES: Three foals, aged between 5 and 10 days, were presented for assessment of lethargy, abdominal pain and joint effusion. Fibrinous pericarditis and pericardial effusion (PE) were recognised in each foal and considered as sequelae to systemic inflammatory response syndrome (SIRS) and suspected or confirmed septicaemia. CLINICAL COURSE AND OUTCOME: Diagnosis of pericarditis was made in two foals by echocardiographic examination and analysis of pericardial fluid, and during postmortem examination of the third foal. In both of the foals that underwent pericardiocentesis, PE was an exudate, no bacteria were identified on cytological analysis and bacterial culture was negative. Despite apparent response to treatment, two foals died 2 and 3 weeks, respectively, after discharge from hospital. One foal was euthanased during hospitalisation. CONCLUSION AND CLINICAL RELEVANCE: This report highlights the need to consider the development of pericarditis and PE in foals with SIRS and signs of cardiorespiratory dysfunction, and the requirement for protracted follow-up to monitor for clinical resolution.

Combination fibrinolytic therapy in the treatment of chronic septic pleuropneumonia in a Thoroughbred gelding.

This report documents the treatment of a case of chronic pleuropneumonia in a 3-year-old Thoroughbred gelding. A recombinant tissue plasminogen activator (tenecteplase) and a recombinant deoxyribonucleic acidase (alphadornase) were infused into the pleural cavity as adjunctive therapy in the early stages of treatment. Instillation of fibrinolytic drugs was associated with a subjective reduction in the amount of fibrin deposition and decreased fluid accumulation within the pleural cavities. Fibrinolytic therapy may be a useful adjunctive therapy in selected cases of intrapleural disease in horses.

Prevalence and distribution of meticillin-resistant Staphylococcus aureus within the environment and staff of a university veterinary clinic.

OBJECTIVES: To characterise the distribution of meticillin-resistant Staphylococcus aureus within the environment of a university small animal hospital and compare this with the distribution among staff. METHODS: Samples were collected from 140 environmental sites and the anterior nares of 64 staff members at the University of Glasgow Small Animal Hospital on a single day (d1). Sixty of the environmental sites were resampled 14 days later (d14). RESULTS: Meticillin-resistant S aureus was isolated from two of 140 (1.4 per cent; 95 per cent confidence interval: 1.7 to 5.1) environmental sites on d1 and one of 60 (1.7 per cent; 95 per cent confidence interval: 0.4 to 8.9) on d14. Two of the 64 staff sampled were positive for meticillin-resistant S aureus (3.1 per cent; 95 per cent confidence interval: 0.4 to 8.4). CLINICAL SIGNIFICANCE: A lower prevalence of meticillin-resistant S aureus was observed in the environment than previously reported. The location, relatedness between isolates and the presence of Panton-Valentine leucocidin indicates that the source of the environmental meticillin-resistant S aureus was most likely to have been human rather than animal in these cases. This study presents important information regarding the potential source and distribution of meticillin-resistant S aureus within veterinary hospital environments and highlights potential variability of prevalence of meticillin-resistant S aureus within and between veterinary institutions.

The Bordetella bhu locus is required for heme iron utilization.

Bordetella pertussis and Bordetella bronchiseptica are capable of obtaining iron from hemin and hemoglobin. Genes encoding a putative bacterial heme iron acquisition system (bhu, for Bordetella heme utilization) were identified in a B. pertussis genomic sequence database, and the corresponding DNA was isolated from a virulent strain of B. pertussis. A B. pertussis bhuR mutant, predicted to lack the heme outer membrane receptor, was generated by allelic exchange. In contrast to the wild-type strain, bhuR mutant PM5 was incapable of acquiring iron from hemin and hemoglobin; genetic complementation of PM5 with the cloned bhuRSTUV genes restored heme utilization to wild-type levels. In parallel studies, B. bronchiseptica bhu sequences were also identified and a B. bronchiseptica bhuR mutant was constructed and confirmed to be defective in heme iron acquisition. The wild-type B. bronchiseptica parent strain grown under low-iron conditions produced the presumptive BhuR protein, which was absent in the bhuR mutant. Furthermore, production of BhuR by iron-starved B. bronchiseptica was markedly enhanced by culture in hemin-supplemented medium, suggesting that these organisms sense and respond to heme in the environment. Analysis of the genetic region upstream of the bhu cluster identified open reading frames predicted to encode homologs of the Escherichia coli ferric citrate uptake regulators FecI and FecR. These putative Bordetella regulators may mediate heme-responsive positive transcriptional control of the bhu genes.

Transcriptional activation of Bordetella alcaligin siderophore genes requires the AlcR regulator with alcaligin as inducer.

Genetic and biochemical studies have established that Fur and iron mediate repression of Bordetella alcaligin siderophore system (alc) genes under iron-replete nutritional growth conditions. In this study, transcriptional analyses using Bordetella chromosomal alc-lacZ operon fusions determined that maximal alc gene transcriptional activity under iron starvation stress conditions is dependent on the presence of alcaligin siderophore. Mutational analysis and genetic complementation confirmed that alcaligin-responsive transcriptional activation of Bordetella alcaligin system genes is dependent on AlcR, a Fur-regulated AraC-like positive transcriptional regulator encoded within the alcaligin gene cluster. AlcR-mediated transcriptional activation is remarkably sensitive to inducer, occurring at extremely low alcaligin concentrations. This positive autogenous control circuit involving alcaligin siderophore as the inducer for AlcR-mediated transcriptional activation of alcaligin siderophore biosynthesis and transport genes coordinates environmental and intracellular signals for maximal expression of these genes under conditions in which the presence of alcaligin in the environment is perceived.

Kinetics and mechanism of iron(III) dissociation from the dihydroxamate siderophores alcaligin and rhodotorulic acid.

The kinetics and mechanism of siderophore ligand dissociation from their fully chelated Fe(III) complexes is described for the highly preorganized cyclic tetradentate alcaligin and random linear tetradentate rhodotorulic acid in aqueous solution at 25 degrees C (Fe2L3 + 6H+ reversible 2 Fe3+ aq + 3 H2L). At siderophore:Fe(III) ratios where Fe(III) is hexacoordinated, kinetic data for the H(+)-driven ligand dissociation from the Fe2L3 species is consistent with a singly ligand bridged structure for both the alcaligin and rhodotorulic acid complexes. Proton-driven ligand dissociation is found to proceed via parallel reaction paths for rhodotorulic acid, in contrast with the single path previously observed for the linear trihydroxamate siderophore ferrioxamine B. Parallel paths are also available for ligand dissociation from Fe2(alcaligin)3, although the efficiency of one path is greatly diminished and dissociation of the bis coordinated complex Fe(alcaligin)(OH2)2+ is extremely slow (k = 10(-5) M-1 s-1) due to the high degree of preorganization in the alcaligin siderophore. Mechanistic interpretations were further confirmed by investigating the kinetics of ligand dissociation from the ternary complexes Fe(alcaligin)(L) in aqueous acid where L = N-methylacetohydroxamic acid and glycine hydroxamic acid. The existence of multiple ligand dissociation paths is discussed in the context of siderophore mediated microbial iron transport.

Cross-referenced combinatorial libraries for the discovery of metal-complexing ligands: library deconvolution by LC-MS.

N-Acylthioureas are excellent ligands for a variety of heavy metals, but their metal selectivity is highly dependent on the precise nature of the substituents present. In this paper we show how combinatorial chemistry techniques can be used to establish relative affinities for copper within a mixture of 100 such thioureas. Following a straightforward synthesis, and copper extraction using standard liquid-liquid extraction techniques, LC-MS was used to identify the ligands which bind most strongly to the copper ions. Among the 100 ligands XC(O)N(Z)C(S)NHY, the most important substituent is the Y group bound to the NH: only aromatic Y substituents give strong binding to copper. The acyl X substituents are invariably aromatic, and an electron-rich X group is best; the affinity for copper seems to be less dependent on the Z substituent, although a large group such as benzyl disfavours copper binding. The five ligands from the library which bind copper most strongly have been clearly identified by a series of experiments: they all have aromatic groups in the Y position, but the X and Z substituents can be more varied. This is a very convincing demonstration of the power of combinatorial methods: to have found the same information by conventional methods would have required a lengthy and repetitive series of syntheses and investigations. In addition, our results give some preliminary evidence for synergistic binding of two different ligands, but this requires further investigation.

Essential role of the iron-regulated outer membrane receptor FauA in alcaligin siderophore-mediated iron uptake in Bordetella species.

Phenotypic analysis using heterologous host systems localized putative Bordetella pertussis ferric alcaligin transport genes and Fur-binding sequences to a 3.8-kb genetic region downstream from the alcR regulator gene. Nucleotide sequencing identified a TonB-dependent receptor family homolog gene, fauA, predicted to encode a polypeptide with high amino acid sequence similarity with known bacterial ferric siderophore receptors. In Escherichia coli, the fauA genes of both B. pertussis and Bordetella bronchiseptica directed the production of a 79-kDa polypeptide, approximating the predicted size of the mature FauA protein. B. bronchiseptica fauA insertion mutant BRM17 was unable to utilize ferric alcaligin, and in complementation analyses ferric alcaligin utilization was restored to this mutant by supplying the wild-type fauA gene in trans. Mutant BRM18, carrying a nonpolar in-frame fauA deletion mutation, was defective in ferric alcaligin utilization and (55)Fe-ferric alcaligin uptake and no longer produced a 79-kDa iron-regulated outer membrane protein. In complementation analyses, BRM18 merodiploids bearing the wild-type fauA gene in trans regained ferric alcaligin siderophore transport and utilization functions and produced the 79-kDa protein. Analysis of a plasmid-borne fauA-lacZ operon fusion confirmed that fauA is subject to iron regulation at the transcriptional level and that cis-acting transcriptional control elements mediating fauA iron repressibility reside within the 3.8-kb PstI fauA DNA region. Moreover, expression of the fauA-lacZ fusion gene under iron starvation conditions was shown to be alcR dependent. FauA is a 79-kDa iron-regulated outer membrane receptor protein required for transport and utilization of ferric alcaligin siderophore complexes by Bordetella species.

Transcriptional analysis of the Bordetella alcaligin siderophore biosynthesis operon.

The alc gene cluster of Bordetella pertussis includes three genes, alcA, alcB, and alcC, which are involved in alcaligin siderophore biosynthesis in response to iron starvation. The production of AlcA, AlcB, and AlcC in Bordetella cells and the transcriptional organization of alcA, alcB, and alcC were investigated by using a set of three alc'-'lacZ gene fusion constructs that were contiguous with the known promoter upstream of alcA and extended to fusion junctions within each alc cistron. All three alc'-'lacZ fusions exhibited iron-repressible reporter gene expression which was abolished by deletion of the 105-bp alcA promoter-operator region. In an immunoblot analysis using a monoclonal antibody specific for beta-galactosidase, the AlcA-LacZ, AlcB-LacZ, and AlcC-LacZ hybrid proteins were detected in Bordetella cells grown under iron-depleted conditions. A B. pertussis mutant in which the 105-bp alcA promoter-operator region was deleted by allelic exchange was unable to produce detectable levels of siderophore. Hybridization analysis using gene-specific probes showed that alc-specific transcript levels in the mutant were negligible compared with those of the wild-type parent. These results confirm that alcA, alcB, and alcC are cotranscribed from an iron-regulated control region immediately upstream of alcA. Transcript analysis using hybridization probes representing regions downstream of alcC demonstrated that alc transcription extends approximately 3.6 kb further downstream from the alcC coding region, suggesting the cotranscription of additional, uncharacterized alcaligin system genes.

Identification and characterization of alcR, a gene encoding an AraC-like regulator of alcaligin siderophore biosynthesis and transport in Bordetella pertussis and Bordetella bronchiseptica.

A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels. In-frame alcR deletion mutants of B. pertussis and B. bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned alcR gene provided in trans restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.

Colicins B and Ia as novel counterselective agents in interspecies conjugal DNA transfers from colicin-sensitive Escherichia coli donors to other gram-negative recipient species.

Colicins are powerful counterselective agents in conjugal DNA transfers using colicin-sensitive Escherichia coli donor strains and colicin-resistant recipient species. We have constructed a high-level constitutive expression system for the bacteriocin and immunity genes of channel-forming colicins B and Ia to optimize production of these colicins as molecular biology reagents.

Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes.

Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4.5-kb BamHI-Smal Bordetella pertussis genomic DNA fragment carried multiple genes required to restore alcaligin production to these siderophore-deficient mutants. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mutations were genetically separable from the BRM6 mutation, and both insertions exerted strong polar effects on expression of the downstream gene defined by the BRM6 mutation, suggesting a polycistronic transcriptional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella promoter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragment. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested the presence of three alcaligin system genes, namely, alcA, alcB, and alcC. The deduced protein products of alcA, alcB, and alcC have significant primary amino acid sequence similarities with known microbial siderophore biosynthesis enzymes. Primer extension analysis mapped the transcriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur repressor-binding site and demonstrated iron regulation at the transcriptional level.

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica.

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)- tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone+ ++, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and ere active in [55Fe]ferric alcaligin transport assays. Evidence suggests that several C2-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.

The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin.

Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.

Epitope insertions define functional and topological features of the Escherichia coli ferric enterobactin receptor.

The outer membrane protein FepA of Escherichia coli is the receptor for the ferric enterobactin siderophore complex and colicins B and D. A foreign antigenic determinant inserted into selected FepA sites allowed mutational analysis of receptor function and in situ immunological tracking of specific protein domains with respect to the bacterial cell compartment. Immunoblot analysis of bacterial proteins using an epitope-specific antibody detected the peptide determinant in the receptor fusions. The impact of the insertions on FepA function was examined by ferric enterobactin-mediated iron uptake experiments and colicin sensitivity tests. In all cases, FepA retained biological activity despite introduction of the foreign sequence. To further develop the topological model of FepA, the peptide-specific antibody was used to localize epitope-carrying FepA domains in intact bacterial cells and their isolated membranes. One epitope resided in a region on the exterior of the cell, at the surface of the FepA protein, while other epitopes appeared to be localized to the periplasm or within the outer membrane.

Bordetella pertussis fur gene restores iron repressibility of siderophore and protein expression to deregulated Bordetella bronchiseptica mutants.

We report the isolation and preliminary phenotypic characterization of manganese-resistant Bordetella bronchiseptica mutants with respect to deregulation of siderophore and iron-regulated protein expression. The fur gene of Bordetella pertussis was cloned by genetic complementation of this deregulated phenotype and confirmed as fur by nucleotide sequence analysis.

Isolation and characterization of Bordetella bronchiseptica mutants deficient in siderophore activity.

Iron acquisition by the gram-negative pathogens Bordetella bronchiseptica and Bordetella pertussis is thought to occur by hydroxamate siderophore-mediated transport as well as an apparently siderophore-independent process by which host transferrins bind to bacterial surface receptors. We constructed B. bronchiseptica mutants deficient in siderophore activity by insertional mutagenesis with miniTn5/lacZ1. The mutants could be placed into four distinct complementation groups, as determined from cross-feeding assays which demonstrated restored siderophore synthesis. Mutants deficient in siderophore activity were BRM1, BRM6, and BRM9, exhibiting approximately 36 to 41% of wild-type siderophore levels, and BRM3 and BRM8, which appeared to produce very little or no detectable siderophore. Mutant BRM4 was found to be a leucine auxotroph, while mutants BRM2 and BRM7 could synthesize siderophore only in low-iron medium which was supplemented with various amino acids. Evaluation of all transcriptional fusions revealed an apparent lack of iron-regulated lacZ expression. Genomic regions flanking the transposable element in the siderophore mutants were homologous with B. pertussis chromosomal DNA, while bioassays suggested siderophore cross-feeding between B. pertussis and B. bronchiseptica. These results indicate probable similarity between the siderophore biosynthetic and transport systems of the two species.

Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane.

The ferric enterobactin receptor (FepA) is a high-affinity ligand-specific transport protein in the outer membrane of Gram-negative bacteria. Deletion of the cell-surface ligand-binding peptides of FepA generated mutant proteins that were incapable of high-affinity uptake but that instead formed nonspecific, passive channels in the outer membrane. Unlike native FepA, these pores acted independently of the accessory protein TonB, which suggests that FepA is a gated porin and that TonB acts as its gatekeeper by facilitating the entry of ligands into the FepA channel. The sequence homology among TonB-dependent proteins suggests that all ligand-specific outer membrane receptors may function by this gated-porin mechanism.