Cross protection of a Mannheimia haemolytica A1 Lkt-/Pasteurella multocida ΔhyaE bovine respiratory disease vaccine against experimental challenge with Mannheimia haemolytica A6 in calves.

Bovine respiratory disease causes significant economic losses in both beef and dairy calf industries. Although multi-factorial in nature, the disease is characterized by an acute fibrinous lobar pneumonia typically associated with the isolation of Mannheimia haemolytica. M. haemolytica A1 and A6 are the two most commonly isolated serotypes from cattle, however, the majority of vaccines have not demonstrated cross-serotype protection. In the current study, the efficacy of a novel, attenuated live vaccine, containing both M. haemolytica serotype A1 and Pasteurella multocida, was evaluated in calves challenged with M. haemolytica serotype A6. Although the challenge was more severe than expected, vaccinated calves had reduced clinical scores, lower mortality, and significantly lower lung lesion scores compared to the placebo-vaccinated control group. The results demonstrate that vaccination with an attenuated live vaccine containing M. haemolytica serotype A1 can protect calves against clinical disease following challenge with M. haemolytica serotype A6.

Colostrum from cattle immunized with a vaccine based on iron regulated proteins of Mannheimia haemolytica confers partial protection.

Passive protection afforded by colostrum from cattle vaccinated prepartum with an inactivated combination vaccine against viral pathogens and Mannheimia haemolytica (M. haemolytica) was evaluated against an experimental M. haemolytica challenge. Newborn calves were either fed colostrum from vaccinated dams or control colostrum. At approximately 3 weeks of age 24 calves were experimentally infected with M. haemolytica. Animals of both groups displayed clinical signs of respiratory disease and lung damage. The survival rate was considerably higher in calves which received colostrum from vaccinated cows. Colonies consistent with M. haemolytica were recovered in large numbers from all animals, but the geometric mean recovery was more than ten-times lower in the vaccinate colostrum fed animals. It can be concluded that maternal antibodies partly protected the calves against a severe M. haemolytica challenge.

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.

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.

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.

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.

Constitutive septal murein synthesis in Escherichia coli with impaired activity of the morphogenetic proteins RodA and penicillin-binding protein 2.

The pattern of peptidoglycan (murein) segregation in cells of Escherichia coli with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the D-cysteine-biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. Höltje, and H. Schwarz, J. Bacteriol. 179:2823-2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes, pbpA and rodA, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of rodA mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564-2567, 1998).

Co-ordinate regulation of the Escherichia coli cell cycle or The cloud of unknowing.

A discussion of some aspects of the regulation of chromosome replication, segregation and cell division in Escherichia coli.

Transcription of essential cell division genes is linked to chromosome replication in Escherichia coli.

Cell division normally follows the completion of each round of chromosome replication in Escherichia coli. Transcription of the essential cell division genes clustered at the mra region is shown here to depend on continuing chromosomal DNA replication. After chromosome replication was blocked by either nalidixic acid treatment or thymine starvation, the transcription of these cell division genes was repressed significantly. This suggests a way in which cell division is controlled by chromosome replication.

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.

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.

All major regions of FtsK are required for resolution of chromosome dimers.

Resolution of chromosome dimers, by site-specific recombination between dif sites, is carried out in Escherichia coli by XerCD recombinase in association with the FtsK protein. We show here that a variety of altered FtsK polypeptides, consisting of the N-terminal (cell division) domain alone or with deletions in the proline-glutamine-rich part of the protein, or polypeptides consisting of the C-terminal domain alone are all unable to carry out dif recombination. Alteration of the putative nucleotide-binding site also abolishes the ability of FtsK to carry out recombination between dif sites.

Conservation of gene order amongst cell wall and cell division genes in Eubacteria, and ribosomal genes in Eubacteria and Eukaryotic organelles.

Comparison of genome sequences from Eubacteria and Eukaryotic organelles shows that the order of genes in gene clusters encoding certain highly conserved cell division proteins and ribosomal proteins is itself highly conserved. Experiments with a cluster of cell division and related genes of E. coli have shown that this gene order is not essential for function. Comparisons between genomes also show that no pair of genes are necessarily adjacent in all genomes. The reason for the extreme conservation of order is therefore unknown, although one possible explanation might be the lateral exchange of tightly-linked groups of genes coding for co-adapted sets of proteins.

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.

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.

The cytoplasmic domain of FtsK protein is required for resolution of chromosome dimers.

Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site-specific recombination at the dif locus of Escherichia coli. We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition.

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.

mraY is an essential gene for cell growth in Escherichia coli.

The synthesis of the murein precursor lipid I is performed by MraY. We have shown that mraY is an essential gene for cell growth. Cells depleted of MraY first swell and then lyse. The expression of mraY DNA in vitro produces a 40-kDa polypeptide detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.