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John F Prescott

Publications and source records attributed to John F Prescott.

14 recordsLinked to original sources
PubMed2004

The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.

An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.

Actinomycetales Infections↗
PubMed2003

Analysis of virulence plasmid gene expression of intra-macrophage and in vitro grown Rhodococcus equi ATCC 33701.

Rhodococcus equi is a soil organism that infects macrophages of foals and immunocompromised humans. Virulence in foal isolates is tightly associated with an 80kb plasmid, which includes a pathogenicity island (PI) with a virulence-associated gene family, vap. A DNA microarray containing 66 of 69 putative open reading frames (ORFs) of the virulence plasmid was developed. Virulence plasmid gene expression of R. equi grown in macrophages or under different conditions in vitro was compared against in vitro growth at 30 degrees C, pH=7. When grown in macrophages, all seven vap family genes as well as six ORFs within, but not outside, the PI were induced. Cluster analysis of the gene expression matrix assembled from different growth conditions suggested that those genes that actively responded to environmental changes divided broadly into two groups. One group, orf1, 2, 5, 6-8, 12-15, 19, and 20 (which includes all the vap genes), was induced at 37 degrees C, mostly by low iron, and to a lesser extent by the synergy of low calcium and pH=5. The second group, orf3, 9, and 10, was induced at 37 degrees C by magnesium depletion (produced by EDTA treatment of growth medium). Temperature (37 degrees C) was the most important factor inducing gene expression for the both groups. Iron restriction led to down-regulation of Group II genes and magnesium restriction led to down-regulation of Group I genes. A putative consensus IdeR binding site was identified upstream of vapA, suggesting that vapA is a member of an IdeR regulon in R. equi. Expression of genes inside macrophages was most closely but not completely mimicked by growth of bacteria at 37 degrees C, pH=5, under conditions of restricted iron, calcium and magnesium; that is, similar to environmental factors found inside macrophages.

Animals↗
PubMed2003

Molecular genetic methods in the veterinary clinical bacteriology laboratory: current usage and future applications.

In the last 5 years, numerous molecular methods have been published for the detection and characterization of bacteria in the field of veterinary medicine. PCR has been the most commonly used technology. Although not currently used for clinical veterinary diagnosis, new technologies such as liquid-phase hybridization, real-time PCR, pathogen load determination and DNA/protein microarray have been described and have many possible applications in the clinical bacteriology laboratory because of their sensitivity and efficiency. This review describes the basic principles and application of recently published DNA-based molecular techniques for the purpose of veterinary clinical bacteriological diagnosis. It covers advances in probe hybridization technology, DNA/RNA amplification techniques and other molecular detection methods, including 16S rRNA analysis for bacterial characterization and DNA microarrays for bacterial detection. The review briefly summarizes the application of molecular methods for the diagnosis of specific important bacterial infections of animals, and for other animal pathogens that are slow or difficult to isolate in the clinical bacteriology laboratory. In addition, the molecular detection of antimicrobial resistance genes and of bovine mastitis pathogens is briefly described and current commercially available tests are listed.

Animals↗
PubMed2003

Performance of five serological assays for diagnosis of Rhodococcus equi pneumonia in foals.

The performance of four enzyme-linked immunosorbent assays (ELISAs) (ELISA-6939, ELISA-33701, ELISA-VapA, and ELISA-California) and an agar gel immunodiffusion test for diagnosis of Rhodococcus equi pneumonia in foals was evaluated. Antibody concentrations of foals with culture-confirmed R. equi pneumonia (n = 41) were compared to those of age-matched pasturemates that remained clinically healthy during the entire breeding season (n = 24). For each serological assay evaluated, selection of a low cutoff resulted in high sensitivity but low specificity. Increasing the cutoff value resulted in better specificity but to the detriment of sensitivity. The best diagnostic performance was achieved with ELISA-California at a cutoff of 40%, resulting in a sensitivity of 59% and a specificity of 88%. We conclude that current serological assays do not differentiate between diseased and clinically healthy foals.

Actinomycetales Infections↗
PubMed2003

Immunoglobulin G subisotype responses of pneumonic and healthy, exposed foals and adult horses to Rhodococcus equi virulence-associated proteins.

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised humans. Replication of virulent isolates within macrophages correlates with the presence of a large plasmid which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH), whose functions are unknown. Although cell-mediated immunity is thought to be crucial in eliminating R. equi infection, antibody partially protects foals. The antibody response to both VapA and VapC was similar in six adult horses and six naturally exposed but healthy foals, as well as in eight foals with R. equi pneumonia. The immunoglobulin G (IgG) subisotype response of pneumonic foals to Vap proteins was significantly IgGb biased and also had a trend toward higher IgGT association compared to the isotype association of antibody in adult horses and healthy exposed foals. This suggests that in horses, IgGb and IgGT are Th2 isotypes and IgGa is a Th1 isotype. Furthermore, it suggests that foals which develop R. equi pneumonia have a Th2-biased, ineffective immune response whereas foals which become immune develop a Th1-biased immune response. Pneumonic foals had significantly more antibody to VapD and VapE than did healthy exposed foals. This may indicate a difference in the expression of these two Vap proteins during persistent infection. Alternatively, in pneumonic foals the deviation of the immune response toward VapD and VapE may reflect a bias unfavorable to R. equi resistance. These data indicate possible age-related differences in the equine immune response affecting Th1-Th2 bias as well as antibody specificity bias, which together favor the susceptibility of foals to R. equi pneumonia.

Age Factors↗
PubMed2003

A fluoroquinolone induces a novel mitogen-encoding bacteriophage in Streptococcus canis.

This study investigated whether the recently recognized emergence of canine streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) might be partly attributed to the use of fluoroquinolones to treat Streptococcus canis infections in dogs. Both mitomycin and the fluoroquinolone enrofloxacin caused bacteriophage-induced lysis of S. canis strain 34, an isolate from a case of canine STSS and NF. Fluoroquinolone-evoked, bacteriophage-induced lysis occurred over a range of concentrations similar to those that would occur after treatment of dogs with these agents. To search for a possible bacteriophage-encoded streptococcal superantigen gene(s), a library of the 36.5 (+/-1.1)-kb bacteriophage, designated phisc1, was made by ligating 3- to 7-kb Tsp5091-digested phisc1 fragments into an EcoRI-digested lambdaZapII vector. Recombinants were screened for mitogenic activity by using canine peripheral blood lymphocytes. Of 800 recombinants screened, 11 recombinants with mitogenic effects were identified, and their inserts were sequenced. The highest homology of 11.6 kb of sequenced phisc1 DNA was to the completely sequenced Streptococcus pneumoniae bacteriophage MM1. Seven of the 11 phisc1 sequenced inserts contained a 552-bp open reading frame, scm, with 27% amino acid similarity to pokeweed (Phytolacca americana) mitogen. PCR showed this gene to be present in 22 of 23 S. canis isolates tested. Quantitative reverse transcription-PCR showed that bacteriophage induction was associated with a 58-fold enhancement of expression of this gene relative to that in a noninduced culture of a similar age. The presence of this gene on a fluoroquinolone-induced bacteriophage may explain the association observed between fluoroquinolone use in dogs and the development of canine STTS and NF.

Amino Acid Sequence↗
PubMed2003

Virulence of Rhodococcus equi isolated from cats and dogs.

Nine cat isolates and nine dog isolates of Rhodococcus equi from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent R. equi strains and contained no virulence plasmids.

Animals↗
PubMed2003

16S ribosomal RNA sequence-based identification of veterinary clinical bacteria.

This study evaluated 16S rRNA gene sequence analysis methods as tools for identification of 22 phenotypically difficult to identify veterinary clinical bacterial isolates in a veterinary diagnostic laboratory. The study compared 16S rRNA gene sequencing and conventional phenotypic identification methods. Using 16S rRNA full-gene sequencing, 95% (21/22) of the isolates were identified to the genus level and 86% (19/22) to the species level. The conventional or commercially available manual identification phenotypic characterization methods presumptively identified 91% (20/22) of the isolates to the genus level and 1 isolate to the species level. However, only 55% (12/22) or 4.5% (1/22) of the phenotypic identifications were correct at the genus or species level when they were compared with the 16S rRNA full-gene sequencing. This study also compared 16S rRNA full-gene and partial-gene sequencing. The results demonstrated that the best 16S rRNA gene-sequencing approach is full-gene sequencing because it gives the most precise species identification. Sequencing of the variable regions 1, 2, and 3 of the 16S rRNA gene could be used for tentative identification because the ability of this sequencing to identify bacteria to the genus level is similar to that of the 16S rRNA full-gene sequencing. This method identified only 14% (3/22) isolates differently to the species level compared with the 16S rRNA full gene sequence. Sequencing of the variable regions 7, 8, and 9 is not recommended because it gives more ambiguous identifications. The cost of a 16S RNA full-gene-sequencing analysis was Can 160 dollars and Can 60 dollars for a partial 16S rRNA gene sequence, i.e., sequencing of variable regions 1, 2, and 3 or variable regions 7, 8 and 9.

Bacteria↗
PubMed2002

A novel lipoarabinomannan from the equine pathogen Rhodococcus equi. Structure and effect on macrophage cytokine production.

Rhodococcus equi is a major cause of foal morbidity and mortality. We have investigated the presence of lipoglycan in this organism as closely related bacteria, notably Mycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants. The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and (1)H and (31)P and two-dimensional heteronuclear nuclear magnetic resonance studies. Key structural features of the lipoglycan are a linear alpha-1,6-mannan with side chains containing one 2-linked alpha-d-Manp residue. This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor. In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal alpha-d-Araf residue capping the 2-linked alpha-d-Manp. The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-alpha-d-Manp residues. We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R. equi. These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R. equi infection. These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R. equi.

Animals↗
PubMed2002

Antimicrobial drug use and resistance in dogs.

Fifteen years (1984-1998) of records from a Veterinary Teaching Hospital were analyzed to determine whether antimicrobial drug resistance in coagulase-positive Staphylococcus spp. (S. aureus, S. intermedius) isolated from clinical infections in dogs has increased, and whether there has been a change in the species of bacteria isolated from urinary tract infections in dogs. In coagulase-positive Staphylococcus spp., a complex pattern showing both increases and decreases of resistance to different classes of antimicrobial drugs was observed, reflecting the changing use of different antimicrobial drug classes in the hospital over a similar period (1990-1999). In canine urinary tract infections identified from 1984 to 1998, an increase in the incidence of multiresistant Enterococcus spp. was apparent, with marginal increases also in incidence in Enterobacter spp. and in Pseudomonas aeruginosa, both of which, like Enterococcus spp., are innately antimicrobial-resistant bacteria. A survey of directors of veterinary teaching hospitals in Canada and the United States identified only 3 hospitals that had any policy on use of "last resort" antimicrobial drugs (amikacin, imipenem, vancomycin). Evidence is briefly reviewed that owners may be at risk when dogs are treated with antimicrobial drugs, as well as evidence that some resistant bacteria may be acquired by dogs as a result of antimicrobial drug use in agriculture. Based in part on gaps in our knowledge, recommendations are made on prudent use of antimicrobial drugs in companion animals, as well as on the need to develop science-based infection control programs in veterinary hospitals.

Animals↗
PubMed2002

Rhodococcus equi pleuropneumonia in an adult horse.

A 10-year-old warmblood gelding was evaluated for intermittent pyrexia, dullness, weight loss, and progressive respiratory disease. Multifocal necrotic pneumonia and pleuritis due to Rhodococcus equi infection was diagnosed. Case management is discussed, as well as factors that may have led to this rare cause of pleuropneumonia in an adult horse.

Actinomycetales Infections↗
PubMed2002

Resurgence of leptospirosis in dogs in Ontario: recent findings.

A marked increase in leptospirosis in dogs was observed in 2000, part of an increasing trend observed in previous years in Ontario. The highest frequency of seropositive cases occurred from September to December 2000, with the peak in November. Large breed dogs were particularly affected. Clinical and clinicopathological data for 31 dogs admitted between 1998 and 2000 to the Ontario Veterinary College Veterinary Teaching Hospital were analyzed. Major clinical presenting features were acute onset of anorexia, depression, fever, and vomiting. Ninety percent of dogs, on admission, showed biochemical evidence of injury to several organs, notably combinations in the order of kidney, muscle, pancreas, and liver. Almost all dogs showed increased serum urea and creatinine levels, and the majority had increased total creatine kinase, bilirubin, alkaline phosphatase, and leukocytosis with neutrophilia. One-third were thrombocytopenic. Of dogs with liver-related abnormalities, most had evidence of cholestasis, with or without hepatocellular damage. Based on serologic studies, in the year 2000, the major serovar involved was autumnalis, but bratislava, grippotyphosa, and pomona were also implicated. The microscopic agglutination test often gave a confusing pattern of reactivities to the serovars that were tested. The high reactivity to serovar autumnalis may represent an erroneous or "paradoxical" reaction typical of early leptospiral serology. The year 2000 was the warmest in Ontario in each of the 4 fall months (September-December) of the previous decade, as well as being the third wettest in the fall period in the last decade. The increase in canine leptospirosis, therefore, may, in part, reflect climate change. The number of positive cases declined in 2001 by about one-third of those in 2000, but the number of submissions of sera for diagnosis increased markedly over previous years. Further work is required to isolate and to identify definitively serovars involved in resurgent canine leptospirosis and the common sources for dogs.

Agglutination Tests↗
PubMedDate not supplied

Rhodococcus equi.

Rhodococcus equi is an important cause of subacute or chronic abscessating bronchopneumonia of foals up to 3-5 months of age. It shares the lipid-rich cell wall envelope characteristic of the mycolata, including Mycobacterium tuberculosis, as well as the ability of pathogenic members of this group to survive within macrophages. The possession of a large virulence plasmid in isolates recovered from pneumonic foals is crucial for virulence. The plasmid contains an 27 kb pathogenicity island (PI) that encodes seven related virulence-associated proteins (Vaps), including the immunodominant surface-expressed protein, VapA. Only PI genes are differentially expressed when the organism is grown in macrophages in vitro. Ten of the PI genes, including six Vap genes, have signal sequences, suggesting that they are exported from the cell to interact with the macrophage. Different PI genes are regulated by temperature, pH, iron, oxidative stress and probably also by magnesium, all environmental changes encountered after environmental R. equi are inhaled in dust and are ingested into macrophages in the lung. The basis of pathogenicity of R. equi is its ability to multiply in and eventually to destroy alveolar macrophages. Infectivity is largely or exclusively limited to cells of the monocyte-macrophage lineage. Current evidence suggests that infection of foals with virulent R. equi results in some foals in subversion of cell-mediated immunity and development of an ineffective and sometimes lethal Th2-based immune response. Significant progress has been made recently in the development of R. equi-E. coli shuttle vectors, transformation and random and site specific mutagenesis procedures, all of which will be important in molecular dissection of the mechanisms by which R. equi subverts normal macrophage killing mechanisms and cell-mediated immunity.

Actinomycetales Infections↗