Determination of the duration of antibacterial efficacy following administration of gamithromycin using a bovine Mannheimia haemolytica challenge model.

The antibacterial efficacy of gamithromycin administered once 1, 5, or 10 days prior to a challenge infection with Mannheimia haemolytica serotype A1 was evaluated. Forty calves were randomly allocated on day -11, restricted by body weight, to one of three treatment groups given gamithromycin at 6 mg/kg of body weight 10, 5, or 1 days before challenge or to an untreated control group. M. haemolytica A1 challenge infections were induced on day 0 by depositing 7.4 × 10(7) CFU at the bifurcation of the main bronchus using a bronchoscope. Clinical observations were made daily from the day of allocation to day 10, when necropsy was scheduled; three calves died or were euthanized in extremis on welfare grounds prior to scheduled necropsy. At necropsy the lungs were removed, pneumonic lesions were scored, and samples of lung tissue were cultured for M. haemolytica. The three groups of animals treated with gamithromycin before challenge had significantly lower lung M. haemolytica counts and fewer clinical signs of respiratory disease than did the saline-treated group. For most of the clinical parameters, the pattern of responses differed significantly (P < 0.05) between the gamithromycin-treated groups and the control group. There were no statistically significant differences between groups in the mean lung lesion scores, partly as a result of high individual variability, particularly within the control group. The administration of gamithromycin 1, 5, and 10 days prior to M. haemolytica A1 challenge resulted in a reduction in bacterial isolation from the lungs and a reduction in the severity of clinical disease.

What is the minimum number of dedicated functions required for a basic cell cycle?

The genome of Escherichia coli has a coding capacity for about 4500 proteins but only a small number of these appear to be specific for the periodic events (initiation of DNA replication, chromosome partitioning and cell division) that punctuate the cell-duplication cycle: furthermore, many of these cell cycle dedicated functions are dispensible under certain conditions, although their presence undoubtedly increases the fitness of the organism to survive in a competitive environment. A simplified but effective cell replication cycle can probably operate with only a few cycle-dedicated proteins, in addition to those required for cell growth itself.

Structure and function of the ftsH gene in Escherichia coli.

The ftsH mutant Y16 shows thermosensitive filamentation with reduced amounts of penicillin-binding protein 3 (PBP3) (Ferreira et al., 1987). Genetic analysis, however, showed that the lethality of the ftsH mutation was not due to a lack of PBP3 activity alone. The ftsH gene was cloned and sequenced and the FtsH protein was deduced to be a membrane protein of 70.7 kDa which has an ATP-binding domain. Highly significant homology of amino acid sequence was observed between FtsH protein and two eukaryotic proteins, yeast Saccharomyces cerevisiae Sec 18p and its mammalian homologue NSF, which are involved in protein transport pathways. This suggests that FtsH protein may act for translocation of specific proteins including PBP3 and at least one other additional protein essential for cell growth. Suppressor mutants of Y16, which were able to grow at 42 degrees C, were isolated, and the suppressor mutations (sfh) were mapped to 16 min. A wild type chromosomal fragment able to complement the sfh mutations was cloned. We also identified another gene (ftsJ) affecting cell division in the region upstream of the ftsH gene.

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.

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.

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.

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.

Characterisation of ovine Bordetella parapertussis isolates by analysis of specific endotoxin (lipopolysaccharide) epitopes, filamentous haemagglutinin production, cellular fatty acid composition and antibiotic sensitivity.

Isolates of Bordetella parapertussis, recovered from sheep or man, were characterised by reaction with specific anti-Bordetella lipopolysaccharide monoclonal antibodies, production of filamentous haemagglutinin, fatty acid patterns, and antibiotic sensitivity. Generally, the isolates lay within one of four groups, with separation of the ovine isolates into two groups. Reactions with specific monoclonal antibodies against lipopolysaccharide separated the ovine isolates into these two groupings. Analysis of the cellular fatty acid compositions by cluster analysis differentiated between the human and the ovine strains and also showed variation within the ovine isolates. When the production of filamentous haemagglutinin was analysed in an ELISA system, a similar pattern emerged. Varying concentrations of filamentous haemagglutinin (11-429 ng (mg total protein)-1) were extracted from the human isolates and the one group of ovine isolates with no significant protein detected in the other ovine group. These studies demonstrate variation between and within B. parapertussis isolates recovered from two mammalian sources.

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.

Electronmicroscopy of the surface of Pasteurella haemolytica.

The surfaces of Pasteurella haemolytica, biotype A, serotypes 1, 2, 6, 7 and 9 and of P. haemolytica, biotype T serotypes 3, 4, 10 and 15 were examined by transmission electronmicroscopy with ruthenium red staining and polycationic ferritin labelling, by scanning electronmicroscopy, and by light microscopy. Electronmicroscopy showed that the surface of strains of P. haemolytica biotype A was covered by irregular protrusions which were probably capsular material. The surface and general morphology of P. haemolytica biotype T were distinct from those of biotype A.

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.

Cytotoxic effect of serotypes of Pasteurella haemolytica on sheep bronchoalveolar macrophages.

Ovine isolates of the 15 known serotypes found within the A and T biotypes of Pasteurella haemolytica were cytotoxic for sheep bronchoalveolar macrophages (BAM). Weaker toxicity for the same target cells was also expressed by non-serotypable ovine isolates of P. haemolytica. The results suggest that cytotoxicity for sheep BAM is a virulence factor common to both A and T biotypes of P. haemolytica.

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.

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.

Specificity of the enzyme-linked immunosorbent assay (ELISA) for antibodies in the sera of specific pathogen-free lambs vaccinated with Pasteurella haemolytica antigens.

Pooled serum from specific pathogen-free (SPF) lambs vaccinated with sodium salicylate extracted (SSE) antigens of Pasteurella haemolytica serotype A1 was shown to contain antibody to other A serotype SSE antigens when tested by the enzyme-linked immunosorbent assay (ELISA). Specific antibody to serotype A1 SSE antigens was demonstrated by absorption of the serum pool with heterologous serotype SSE antigens. The type-specific antigens of serotypes A1 and A9 were prepared by phenol--water extraction (PWE) of their respective SSE antigens. The PWE antigens were examined in a sandwich ELISA where rabbit IgG anti-P. haemolytica A1 cells or A9 cells was used as a coating layer to bind PWE antigens. The specificity of these antigens was demonstrated by marked reduction of reactivity between serum from SPF lambs vaccinated with SSE of serotypes A1 or A9.

A crude cytotoxin vaccine protects sheep against experimental Pasteurella haemolytica serotype A2 infection.

Three vaccines containing Pasteurella haemolytica serotype A2 antigens were tested for their ability to protect sheep against a homologous challenge. A crude cytotoxin preparation in combination with a sodium salicylate extract (SSE) or crude cytotoxin alone were highly protective (98 and 86%, respectively), whereas SSE alone was poorly (47%) protective. These findings indicated that the crude cytotoxin was an essential component of a protective vaccine. Protection correlated with serum cytotoxin-neutralising (CN) titres and bactericidal activity, which were stimulated by antigens in the crude cytotoxin preparation.

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.

Protection of lambs against experimental pneumonic pasteurellosis by transfer of immune serum.

Passive protection of specific pathogen-free lambs against experimental pasteurellosis was achieved using antisera from conventionally reared sheep which were either convalescent from experimental pneumonia or inoculated with Pasteurella haemolytica A2 vaccines. The complete immune sera, or immunoglobulin-rich fractions prepared from them, when administered separately or together provided 94-100% protection of recipients compared to control lambs. Antibodies to P. haemolytica in donor sera were quantified by anti-sodium salicylate extract (SSE) and anti-lipopolysaccharide (LPS) ELISA, bactericidal assay, cytotoxin neutralization and indirect haemagglutination. The anti-SSE ELISA titres correlated best with protective efficacy and could be used to measure antibody in recipient lambs immediately before challenge. The degree of protection was unaffected by prior infection with parainfluenza virus Type 3, suggesting that such exposure did not enhance exudation of circulating immunoglobulin into the respiratory tract. It was concluded that systemic humoral immunity alone can prevent pasteurellosis.

Bronchopneumonia in mice caused by Pasteurella haemolytica A2 after predisposition by ovine Bordetella parapertussis.

Initial intranasal inoculation of four to eight-week-old Swiss White mice with 7.5 x 10(6) colony forming units (cfu) of ovine B. parapertussis followed 30 min, three or five days, by intranasal inoculation with 1.4 x 10(5) cfu of Pasteurella haemolytica A2 resulted in a more severe infection pattern than when either agent was administered alone. Histopathological examination showed that inoculation with B. parapertussis alone caused a bronchopneumonia the severity of which was dependant upon the infecting dose. Bacteria were recovered up to 10 days after inoculation. P. haemolytica alone had no apparant pathogenic effect and was cleared from the lungs within 24 h. When both agents were given in combination the lesions were most severe when P. haemolytica was administered three days after B. parapertussis infection. These findings suggest that B. parapertussis predisposes mice to subsequent infection with P. haemolytica and that the timing of the P. haemolytica administration influences the severity of the lung lesions.

Immunity of specific pathogen-free lambs to challenge with an aerosol of Pasteurella haemolytica biotype A serotype 2. Pulmonary antibody and cell responses to primary and secondary infections.

Specific pathogen-free (SPF) lambs previously exposed to an aerosol of P. haemolytica biotype A serotype 2 (A2) were immune to subsequent challenge with an aerosol of P. haemolytica A2. Untreated control lambs were not immune to this challenge. The local immune responses of the lung to these challenges were examined. High IgG and IgA titres to P. haemolytica and high levels of opsonizing antibody against P. haemolytica were present in the lung washings from previously infected immune lambs at autopsy, seven days after the second infection. Lung washings from control lambs, 7 days after challenge with P13 virus and P. haemolytica A2, had no IgG titres, very little opsonizing activity but did have IgA titres which were significantly higher than in unchallenged control lambs. The cellular response of animals challenged with P13 virus and P. haemolytica was significantly greater than that of unchallenged controls or of lambs exposed only to P. haemolytica. However, this finding was complicated by the response to P13 virus. Lymphocytes from lung washings of all lambs failed to respond in a lymphocyte stimulation test to phytohaemagglutinin while blood lymphocytes did respond. There was little specific response to P. haemolytica antigen in the test.