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Biomedical subjects

W Donachie

Publications and source records attributed to W Donachie.

At least 19 recordsLinked to original sources

Experimental induction of pneumonic pasteurellosis in calves by intratracheal infection with Pasteurella multocida biotype A:3.

The study aimed to establish an experimental model to investigate the pathogenesis of lung infection by Pasteurella multocida, an important cause of bovine respiratory disease. An experimental model is required to assist the development of an effective vaccine. Sixteen 8-week-old calves were challenged intratracheally with 10(9) or 10(10) colony forming units of P. multocida in either 60 or 300 ml saline in a 2 x 2 factorial experiment. All animals became dull within 2-6h post-infection (p.i.) and two calves were killed humanely because of suspected endotoxic shock. Remaining animals showed increased respiratory rates by 15-20 h p.i. and, at 23 h p.i., calves given the high dose, high volume challenge showed higher (P < 0.05) rectal temperatures. From 24 to 36 h p.i., clinical signs decreased in a majority of animals. Plasma haptoglobin concentrations increased (P < 0.05) in calves given the high volume challenge irrespective of the number of bacteria. At post-mortem examination (4d p.i.), lung lesions, mainly in the apical lobes, were found in all calves. Histopathological examination showed areas of purulent pneumonia with a tendency to abscessation and inflamed interlobular septa characterised by accumulation of neutrophils and oedema. The clinical and pathological responses described were typical of bovine pneumonic pasteurellosis.

Animals↗

Typing of Pasteurella multocida isolated from pigs with and without porcine dermatitis and nephropathy syndrome.

Porcine dermatitis and nephropathy syndrome (PDNS) is a sporadic, usually fatal disease of growing and finishing pigs that has been recognized in many pig-producing countries. Pasteurella multocida strains isolated from 15 pigs with PDNS and 51 pigs without PDNS were characterized by capsule and somatic antigen typing, random amplified polymorphic DNA (RAP-D) typing, and restriction analysis of genomic DNA using pulsed-field gel electrophoresis (PFGE). While capsular, somatic, and RAP-D typing did not discriminate PDNS isolates from non-PDNS isolates, all of the isolates from PDNS cases showed an identical ApaI PFGE restriction pattern. This pattern was also found in a high proportion (36%) of P. multocida strains isolated from non-PDNS cases. Isolation of a single variant of P. multocida from tissues of pigs with PDNS warrants further investigation into the possible role of these bacteria in the etiology of the disease.

Animals↗

Survival of Mannheimia (Pasteurella) haemolytica in tracheobronchial washings of sheep and cattle.

The growth, morphology and long-term survival of a representative isolate of Mannheimia haemolytica serotypes A1 and A2 were monitored in ovine and bovine tracheobronchial washings. Both strains survived for at least 244 days in ovine tracheobronchial washings and 156 days in bovine tracheobronchial washings. The addition of fresh washings at these times prompted an increase for serotype A2 but no change in viability for serotype A1 in ovine tracheobronchial washings and an increase for both serotypes in bovine tracheobronchial washings. When growth and survival was compared using tracheobronchial washings from ruminant and non-ruminant species there was a trend towards longer survival in ruminant fluids.Long-term survival was associated with temporary or permanent change from normal size colonies to 'micro-colonies' on sheep blood agar. Subculture allowed reversion to normal colony morphology. Analysis showed these micro-colonies to consist of chains of elongated bacteria. M. haemolytica serotype A2 was more robust in its ability to withstand nutrient deprivation for long periods of time. These survival mechanisms may have important implications for pathogenesis.

Animals↗

Adherence of ovine and human Bordetella parapertussis to continuous cell lines and ovine tracheal organ culture.

The adherence of ovine and human isolates of Bordetella parapertussis to ovine and human continuous culture cell lines and to ovine tracheal organ culture was compared. Adherence to non-ciliated respiratory continuous culture cells did not reveal any host-specificity of the isolates. In contrast, adherence of ovine B. parapertussis strains to ciliated ovine tracheal organ culture was significantly greater than that of human strains. These results indicate that tracheal organ culture is a useful tool for studying host-specific adherence of B. parapertussis and suggest that adherence of B. parapertussis to ciliated epithelia is species-specific making it unlikely that the transfer of B. parapertussis between humans and sheep will result in an infection.

Animals↗

Characterization of United Kingdom isolates of Corynebacterium pseudotuberculosis using pulsed-field gel electrophoresis.

Caseous lymphadenitis is a chronic suppurative disease caused by Corynebacterium pseudotuberculosis and is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates of C. pseudotuberculosis from the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis using SfiI and SmaI. All of the isolates were confirmed as C. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction with SfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.

Animals↗

Investigations on the species specificity of Mannheimia (Pasteurella) haemolytica serotyping.

Eleven serotypes (1, 2, 5-9, 12-14 and 16) have been demonstrated within Mannheimia haemolytica. Subsequent serotyping of 166 Mannheimia haemolytica-like strains, genetically and phenotyphically distinct from Mannheimia haemolytica, and isolated from ruminants, pigs, hares and rabbits showed that 13.2% were typeable, 19 of which were serotype 11 representing strains now being classified as M. glucosida. In addition, three strains belonged to serotypes 6, 9 and 16, respectively. Additionally, the serotyping results of 98 (P.) haemolytica-like isolates from non-ruminant sources collected by the UK Veterinary Investigation Centres during the period 1982-1996 were investigated. None of these isolates have been kept, making further genetic characterization impossible. Among these isolates, 25.5% were typeable representing serotypes 1, 2, 3, 5, 6, 9, 10, 13 and 15. Substantial evidence has been reported indicating that M. haemolytica-like isolates from non-ruminant sources represent species different from M. haemolytica. The present investigation underlines that serotyping does not represent a reliable method for the identification of M. haemolytica or M. glucosida. These observations emphasize that extended phenotypic and genetic characterization is necessary for the proper identification of these organisms.

Animals↗

Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis.

Forty-three Listeria ivanovii isolates were collected in the UK between 1991 and 1997 from: 35 animal infections; two human infections; five foods; and one environmental source. A further two type strains of L. ivanovii (subsp. ivanovii and subsp. londoniensis) were obtained from a culture collection. These bacteria were characterised by conventional phenotypic methods and by pulsed-field gel electrophoresis (PFGE) using ApaI and SmaI. Forty-two of the isolates from the UK were identified as L. ivanovii subsp. ivanovii and the remaining culture as L. ivanovii subsp. londoniensis. Six and four PFGE profiles were obtained using ApaI and SmaI digestion respectively; six composite profiles were obtained combining the results for both enzymes. The PFGE profile of the UK L. ivanovii subsp. londoniensis (isolated from processed shrimps) was similar to the type strain of this subspecies and differed from all of the L. ivanovii subsp. ivanovii tested. The majority of isolates (38 out of 45) belonged to one profile showing that the UK population of this bacterium is much less genetically diverse than similar studies have shown for Listeria monocytogenes.

Animals↗

The use of dummy data points when fitting bacterial growth curves.

We consider the problem of fitting mathematical models for bacterial growth and decline to experimental data. Using models which represent the phases of the growth and decline cycle in a piecewise manner, we describe how least-squares fitting can lead to potentially misleading parameter estimates. We show how these difficulties can be overcome by extending a data set to include hypothetical observations (dummy data points) which reflect biological beliefs, and the resulting stabilization of parameter estimates is analysed mathematically. The techniques are illustrated using real and simulated data sets.

Anaerobiosis↗

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue.

In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasteurella haemolytica grown under iron-depleted conditions. This study reports identification of the gene (fbpA) encoding the 35 kDa protein based on complementation of an entA Escherichia coli strain transformed with a plasmid derived from a P. haemolytica lambda ZAP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated ORF identified on the recombinant plasmid corresponded with the N-terminal amino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding the 35 kDa protein demonstrated homology with the cluster 1 group of extracellular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert identified three other predominant ORFs, two of which appeared to be in an operonic organization with fbpA. These latter components (fbpB and fbpC) showed homology to the transmembrane and ATPase components of ATP-binding cassette (ABC)-type uptake systems, respectively. Based on amino acid/DNA sequencing, citrate competition assay of iron affinity and visible wavelength spectra, it was concluded that the P. haemolytica 35 kDa protein functions as an FbpA homologue (referred to as PFbpA) and that the gene encoding this protein is part of an operon comprising a member of the FbpABC family of iron uptake systems. Primary sequence analysis revealed rather surprisingly that PFbpA is more closely related to the intracellular Mn/Fe-binding protein IdiA found in cyanobacteria than to any of the homologous FbpA proteins currently known in commensal or pathogenic members of the Pasteurellaceae or Neisseriaceae.

Amino Acid Sequence↗

Divergent activity and function of superoxide dismutases in Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10.

Representative strains of Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were examined for the presence of superoxide dismutase. Visualisation of superoxide dismutase enzyme activity on polyacrylamide gels, and specific inhibition with potassium cyanide verified a copper/zinc (Cu/Zn) superoxide dismutase only in serotype A2 whereas serotypes A1 and T10 showed other superoxide dismutase activity. Using a simple freeze-thaw method the cellular location of superoxide dismutase enzyme activity was determined in all three serotypes. In serotypes A1 and A2 but not T10 superoxide dismutases were located in the periplasm. The viability of serotypes A2 and T10 cells in the presence of exogenous superoxide was unchanged over a 30 min period, whereas serotype A1 cells declined in viability between 15 and 30 min. Purified immunoglobulin from sheep convalescent serum did not reduce superoxide dismutase activity in the serotypes in an in vitro assay. The presence of this enzyme within the pasteurellae suggests a supportive role in the virulence of this major pathogen of ruminants.

Animals↗

A review of Listeria monocytogenes and listeriosis.

Following the initial isolation and description in 1926 Listeria monocytogenes has been shown to be of world-wide prevalence and is associated with serious disease in a wide variety of animals, including man. Our knowledge of this bacterial pathogen and the various forms of listeriosis that it causes has until recently been extremely limited, but recent advances in taxonomy, isolation methods, bacterial typing, molecular biology and cell biology have extended our knowledge. It is an exquisitely adaptable environmental bacterium capable of existing both as an animal pathogen and plant saprophyte with a powerful array of regulated virulence factors. Most cases of listeriosis arise from the ingestion of contaminated food and in the UK the disease is particularly common in ruminants fed on silage. Although a number of forms of listeriosis are easily recognized, such as encephalitis, abortion and septicaemia, the epidemiological aspects and pathogenesis of infection in ruminants remain poorly understood. The invasion of peripheral nerve cells and rapid entry into the brain is postulated as a unique characteristic of its virulence, but relevant and practical disease models are still required to investigate this phenomenon. This review offers an up to date introduction to the organism with a description of virulence determinants, typing systems and a detailed account of listeriosis in animals. Experimental and field papers are reviewed and further sections deal with the diagnosis, treatment and control of listeriosis in animals. A final part gives an overview of listeriosis in man.

Animal Diseases↗

An outbreak of Pasteurella multocida pneumonia in lambs during a field trial of a vaccine against Pasteurella haemolytica.

A vaccine against pneumonic pasteurellosis was evaluated for efficacy at two dilutions in lambs transported by sea from New Zealand to Saudi Arabia. The experimental vaccine was a killed Pasteurella haemolytica serotype A1 and A2 preparation. There was no evidence of either dilution of the vaccine leading to a lower pneumonia death or lesion rate than for the control group. However, bacteriological examinations to establish the causality of the pneumonia cases showed Pasteurella multocida to be the dominant organism, while P. haemolytica types A1 and/or A2 occurred at only a very low incidence.

Journal Article↗

Occurrence of [copper, zinc]-cofactored superoxide dismutase in Pasteurella haemolytica and its serotype distribution.

Fifty-two ovine strains of Pasteurella haemolytica and P. trehalosi representing serotypes 1-16 were examined for the presence of [copper, zinc]superoxide dismutase DNA sequences. This was done using a combination of polymerase chain reaction with degenerate primers based on the sequence of the [Cu,Zn]superoxide dismutase gene (sodC) in related species and Southern hybridization using a fragment of sodC from P. haemolytica A2 serotype as a probe. Both detection methods identified a fragment of the sodC gene in 9/9 strains of P. haemolytica serotype 2 examined and in 5/8 strains of serotype 7. No evidence of this gene was found in any other serotype of P. haemolytica or in any P. trehalosi serotype. Comparison of DNA sequence showed near identity between sodC from the A2 and A7 serotypes of P. haemolytica and substantial similarity (70%) to sodC previously sequenced in P. multocida, Haemophilus parainfluenzae and H. influenzae. Analysis by gel electrophoresis of the superoxide dismutase activity present in cell lysates showed that one or more superoxide dismutase is present in all serotypes. However, cyanide-inhibitable activity, corresponding to [Cu,Zn]superoxide dismutase, was detected only in those strains of serotypes A2 and A7 which showed evidence of the sodC gene fragment.

Amino Acid Sequence↗

Differentiation between human and ovine isolates of Bordetella parapertussis using pulsed-field gel electrophoresis.

The genetic relatedness of 18 human and 29 ovine isolates of Bordetella parapertussis was examined by macrorestriction digestion of DNA with the rarely cutting enzyme XbaI and resolution by pulsed-field gel electrophoresis. There was clear separation of human and ovine isolates and variation within host types. The human isolates were separated into three types as were the 24 Scottish ovine isolates. Species-specific bands were observed with the human isolates at 114, 134, 166, 213, 346 and 372 kb. No species-specific bands were found in the B. parapertussis ovine isolates. Isolates of B. parapertussis recovered from sheep in New Zealand gave a further two DNA banding patterns which were clearly different from the Scottish ovine and the human isolates. These results indicate that human and ovine isolates of B. parapertussis are genetically distinct and that variation exists within isolates from the same host species. Pulsed-field gel electrophoresis therefore appears to be a powerful discriminatory tool for the classification of B. parapertussis.

Animals↗

Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates: results of an international comparative study.

Multilocus enzyme electrophoresis (MEE) is a standard technique that is used to elucidate the epidemiology of a variety of bacterial species. Recently, the method has been employed by several laboratories for investigations of clinical and foodborne isolates of Listeria monocytogenes. To assess the sensitivity and reproducibility of MEE in characterising L. monocytogenes isolates for epidemiological purposes and, ultimately, to agree on a standard protocol, seven laboratories participated in a blinded study of 80 strains. The strain collection included both epidemiologically related and unrelated isolates. Each laboratory used its own protocol for MEE. The number of enzymes that were assayed by the laboratories ranged from 8 to 23, and the total number of identified electrophoretic types (ETs) varied between 14 and 25. Of the II pairs of duplicate strains, the number of pairs recognised as identical by the seven laboratories ranged from 3 to 10 (median = 8). From 10 to 18 (median = 15) of the 22 groups of epidemiological related strains were recognised as homogeneous by the different laboratories. The discriminatory power of the method, calculated using Simpson's index of diversity for 69 strains (80 strains minus the 11 duplicates), ranged from 0.827 to 0.925. This relatively low discriminatory power is a consequence of a somewhat low genetic diversity of L. monocytogenes compared to other bacterial species. Efforts should be pursued to standardise the method in order to improve the intra- and inter-laboratory reproducibility.

Alleles↗

Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin.

Defined segments of the leukotoxin A gene (lktA) from an A1 serotype of Pasteurella haemolytica were cloned into a plasmid vector and expressed as LacZ alpha fusion proteins. These fusion proteins were electrophoresed in SDS-PAGE gels and their immunoblotting reactivities with several monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant peptides containing these epitopes were not antigenic, but a polypeptide encompassing 229 amino acids at the C terminus evoked neutralizing antibodies when used to immunize specific-pathogen-free lambs. The distributions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype A1 were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of LktA. Toxin-neutralizing antibodies in convalescent sheep serum were shown to be directed against conformational epitopes by selective absorption of antibodies directed against linear epitopes.

Amino Acid Sequence↗

Intra-specific diversity and host specificity within Pasteurella haemolytica based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins.

Intra-specific diversity within Pasteurella haemolytica was assessed by analysing variation in the capsular polysaccharide (serotypes), lipopolysaccharide (LPS) and outer-membrane proteins (OMPs) of 184 isolates recovered from cattle and sheep. Four 12 serotypes comprised 83% of the total number of isolates, including A1 and A2 as the most frequently recovered serotypes from cattle and sheep, respectively. Nine distinct LPS profiles were identified. Four different core-oligosaccharide patterns were present, each of which occurred alone as rough LPS and also in association with single O-antigen profile as smooth LPS; the ninth LPS type was also smooth but had a different O-antigen profile. The capsular serotypes could be divided into four groups based on the dominant LPS profile within each serotype: (1) A1, A6, A9, A12 and A5; (2) A2, A8, A14 and A16; (3) A7 and A13; and (4) A11. Smooth LPS of type 1A, which was found only in the first group, was associated primarily with bovine disease isolates, whereas rough LPS of types 1B and 3B were associated primarily with group 2 serotypes and ovine disease isolates. Similarly, the variation of OMP profiles generated three groups: (1) A1, A6 A9, A12, A5 and A8; (2) A2, A14 and A16; and (3) A7, A11 and A13. Isolates belonging to groups 2 and 3 exhibited greater diversity in their OMP profiles than those belonging to group 1. Although the majority of group 3 isolates possessed profiles unique to that group, a smaller number of A7 isolates possessed profiles with similarities to those of serotypes A1 or A2. OMP profiles clearly differentiated bovine from ovine isolates of the same serotypes. The association both of specific LPS and OMP profiles with bovine or ovine disease isolates suggested a correlation between specific cell-surface structures and host specificity. The combined analysis of capsular serotypes, LPS types and OMP profiles identified seven major groups within P. haemolytica which were responsible for 59% of the disease cases, suggesting a clonal structure for this species. Overall, comparison of the capsular serotypes, LPS types and OMP profiles proved extremely useful for assessing diversity within P. haemolytica.

Animals↗

The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing.

Listeria monocytogenes was isolated in low numbers from a variety of environmental samples associated with the primary production of food, including vegetation, faeces and meat. The organism was rarely detected on growing grass and vegetables prior to processing. The excretion of L. monocytogenes by farm animals was linked to their diet, with animals fed entirely on hay or manufactured diets not excreting detectable levels of Listeria (i.e. absence in 25 g). However, animals fed on silage, which is frequently contaminated with L. monocytogenes, commonly excreted the organism. Transport of live animals over long distances (> 100 km) significantly increased the level of excretion of Listeria, but the contamination of carcasses of sheep and cattle was not high. Pigs and poultry faeces were free of Listeria prior to slaughter and pig carcasses were not found to have Listeria present. Frozen and chilled chicken did show detectable levels reflecting the greater potential for contamination during poultry processing. Samples of minced beef were tested and 21 of 23 samples were positive for L. monocytogenes, demonstrating that processing significantly increases the level of contamination compared to whole carcasses. Multilocus enzyme electrophoresis of a representative selection of the isolates showed that there was a wide range of electrophoretic types present in the primary production environment, relatively few of which have been linked to cases of human listeriosis. However, these types do arise on farms and occasional contamination of food raw material by potentially virulent strains may be sufficient to allow adaptable strains to become established in the processing environment and thus be responsible for more widespread contamination of the food available to the consumer.

Abattoirs↗