Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.     

The ycf27 genes from cyanobacteria and eukaryotic algae: distribution and implications for chloroplast evolution

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS(M.h.) was able to complement AI-2 production in the Escherichia coli DH5alpha luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.     

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.     

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.     

A minimal medium for growth of Pasteurella multocida

Abstract We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm ). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.     

Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica

Pneumonia is a leading cause of loss to the sheep and cattle industry throughout the world. Mannheimia (Pasteurella) haemolytica is one of the most important respiratory pathogens of domestic ruminants and causes serious outbreaks of acute pneumonia in neonatal, weaned and growing lambs, calves, and goats. M. haemolytica is also an important cause of pneumonia in adult animals. Transportation, viral infections with agents such as infectious bovine rhinotracheitis virus, parainfluenza-3 virus or bovine respiratory syncytial virus, overcrowding, housing of neonates and weaned animals together and other stressful conditions predispose animals to M. haemolytica infection [1, 2]. This review assimilates some of the findings key to cellular and molecular responses of the lung from a pathologist's perspective. It includes some of what is known and underscores areas that are not fully understood.     

Sharing of Pasteurella spp. between free-ranging bighorn sheep and feral goats

Pasteurella spp. were isolated from feral goats and free-ranging bighorn sheep (Ovis canadensis canadensis) in the Hells Canyon National Recreation Area bordering Idaho, Oregon, and Washington (USA). Biovariant 1 Pasteurella haemolytica organisms were isolated from one goat and one of two bighorn sheep found in close association. Both isolates produced leukotoxin and had identical electrophoretic patterns of DNA fragments following cutting with restriction endonuclease HaeIII. Similarly Pasteurella multocida multocida a isolates cultured from the goat and one of the bighorn sheep had D type capsules, serotype 4 somatic antigens, produced dermonecrotoxin and had identical HaeIII electrophoretic profiles. A biovariant U(beta) P.haemolytica strain isolated from two other feral goats, not known to have been closely associated with bighorn sheep, did not produce leukotoxin but had biochemical utilization and HaeIII electrophoretic profiles identical to those of isolates from bighorn sheep. It was concluded that identical Pasteurella strains were shared by the goats and bighorn sheep. Although the direction of transmission could not be established, evidence suggests transmission of strains from goats to bighorn sheep. Goats may serve as a reservoir of Pasteurella strains that may be virulent in bighorn sheep; therefore, goats in bighorn sheep habitat should be managed to prevent contact with bighorn sheep. Bighorn sheep which have nose-to-nose contact with goats should be removed from the habitat.     

Agarose soy casein digest medium for replacement of blood agar for potency determinations of live Pasteurella vaccines

Blood agar, prepared with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) soy agar and 5% defibrinated bovine blood, is used for testing the potency of live Pasteurella multocida and Pasteurella haemolytica vaccines, but its potential for variation makes it undesirable to use in a standard assay method. Tests done with RPMI 1640 and Trypticase soy medium indicated that the benefits obtained by adding defibrinated blood to the Trypticase soy agar medium were more likely due to neutralization of toxic components than to the presence of transferrin or iron as growth factors. Reduction of toxic components in the Trypticase soy agar medium was accomplished by replacing agar with agarose and by autoclaving glucose as a separate solution to produce the replacement medium. The replacement medium was prepared by autoclaving three separate solutions--Trypticase soy broth without glucose; glucose; and agarose--cooling to 55 degrees C, and mixing and then pouring the mixtures into petri dishes. The growth obtained with this medium as judged by determination of the number of CFU and the colony sizes of P. multocida or P. haemolytica was equal to or better than those obtained with blood agar.     

A novel Erm monomethyltransferase in antibiotic-resistant isolates of Mannheimia haemolytica and Pasteurella multocida

Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens.     

Agarose soy casein digest medium for replacement of blood agar for potency determinations of live Pasteurella vaccines.

Blood agar, prepared with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) soy agar and 5% defibrinated bovine blood, is used for testing the potency of live Pasteurella multocida and Pasteurella haemolytica vaccines, but its potential for variation makes it undesirable to use in a standard assay method. Tests done with RPMI 1640 and Trypticase soy medium indicated that the benefits obtained by adding defibrinated blood to the Trypticase soy agar medium were more likely due to neutralization of toxic components than to the presence of transferrin or iron as growth factors. Reduction of toxic components in the Trypticase soy agar medium was accomplished by replacing agar with agarose and by autoclaving glucose as a separate solution to produce the replacement medium. The replacement medium was prepared by autoclaving three separate solutions--Trypticase soy broth without glucose; glucose; and agarose--cooling to 55 degrees C, and mixing and then pouring the mixtures into petri dishes. The growth obtained with this medium as judged by determination of the number of CFU and the colony sizes of P. multocida or P. haemolytica was equal to or better than those obtained with blood agar.     

Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.     

A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia

A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A, P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, 'P. bettii', 'A. seminis', P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as 'BLG', 'Mair', 'Ross' and 'aer-2'. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis, Y. pestis and a group 'ent-b' similar to Y. enterocolitica. Area C contained non-fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group 'past-b'. There were also a small number of unnamed single strains.     

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.     

Investigating the candidacy of lipopolysaccharide-based glycoconjugates as vaccines to combat <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis>

Inner core lipopolysaccharide (LPS) has been shown to be conserved in the majority of veterinary strains from the species Mannheimia haemolytica, Actinobacillus pleuropneumoniae and Pasteurella multocida and as such is being considered as a possible vaccine antigen. The proof-in-principle that a LPS-based antigen could be considered as a vaccine candidate has been demonstrated from studies with monoclonal antibodies raised to the inner core LPS of Mannheimia haemolytica, which were shown to be both bactericidal and protective in a mouse model of disease. In this study we confirm and extend the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against Mannheimia haemolytica wild-type strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes a conjugation strategy that uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule. To protect the amino functionality on the phosphoethanolamine (PEtn) residue of the inner core, we developed a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy with the thiol linker on the carboxyl residues of the carrier protein and the maleimide linker on the carbohydrate resulted in a high loading of carbohydrates per carrier protein. Immunisation derived antisera from rabbits recognised fully extended Mannheimia haemolytica LPS and whole cells from serotypes 1 and 2, despite a somewhat immunodominant response to the linkers also being observed. Moreover, bactericidal activity was demonstrated to a strain elaborating the immunising carbohydrate antigen and crucially to wild-type cells of serotypes 1 and 2, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by Mannheimia haemolytica.     

Aerobic bacteria from nasal discharge of goats suffering from clinical PPR: isolation and identification

Bacteriological isolation of aerobic bacteria from nasal discharge of achondroplastic West African dwarf goats showing clinical signs of peste des petits ruminants (PPR), and the laboratory procedures carried out for the identification of the isolates are described. From a total of forty nasal swabs obtained seventy five isolates were recovered. The percentage frequency of isolation of different organisms in descending order of occurrence is as follows: Staphylococcus 44.0%; Streptococcus 22.67%; Neisseria 12.00%; Pasteurella 10.67%; Pseudomonas 4.00%; Proteus 4.00% and Corynebacterium 1.33%. A further break-down using biochemical tests revealed that Staphylococcus aureus accounted for 30.67% of the total bacteria isolates; Staphylococcus epidermidis 13.33%; Streptococcus viridans 18.67%; Streptococcus pyogenes 4.00%; Neisseria catarrhalis 12.00%; Proteus mirabilis 2.67%; Proteus rettgeri 1.33%; Pseudomonas aeruginosa 5.33%; Pasteurella multocida 4.00%; Pasteurella haemolytica 6.67% and Corynebacterium pyogenes 1.33%.     

Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394-1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5' and 3' ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.     

p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis

To analyse the role of Pasteurella haemolytica Leukotoxin (LKT) in the mechanism of apoptotic cell death of bovine lymphocytes, we evaluated DNA fragmentation and p53 and c-myc expression. P. haemolytica strain ATCC 14003 was cultivated for LKT production. DNA fragmentation was analysed by electrophoresis on Agarose gel. DNA strand breaks in individual apoptotic cells were also detected by an in situ Terminal deoxy nucleotidyl Transferase (TdT). The Polymerase Chain Reaction (PCR) procedure was used for verified p53 and c-myc activation by P. haemolytica LKT. LKT was able to induce DNA fragmentation in a dose and time-dependent fashion. The greatest apoptotic effect was obtained using LKT at a concentration of 0.25 U. The results show that p53 and c-myc activation by LKT is correlated with apoptosis of bovine lymphocytes and monocytes. Our data suggest that LKT may have an important role in the bacterial virulence of Pasteurella haemolytica.     

Pathological and microbiological studies on calf pneumonia occurring in mass rearing facilities

Pathological and microbiological studies were conducted on lesions in the lungs of 194 calves from mass rearing facilities. Macroscopically, the lesions were classified into six forms: nonlesion, atelectasis, mild pneumonia, moderate pneumonia, advanced pneumonia, and advanced pneumonia accompanied with abscess. Histopathological examination revealed bronchopneumonia in most of the calves. Lesions more advanced than moderate pneumonia were complicated with desquamation, severe exudation, and necrosis. Bacteriologically, Pasteurella sp. was isolated often in combination with Staphylococcus sp. from about a half of the atelectatic cases. With the development of pneumonic lesions, Pasteurella sp. was isolated at a high frequency in combination with Haemophilus sp., Streptococcus sp., and Corynebacterium sp. Prominent necrosis was more often seen in cases with Pasteurella haemolytica isolated than in cases with only Pasteurella multocida isolated. Mycoplasma sp. and Ureaplasma sp. were isolated from distinctly pneumonic lesions. Advanced pneumonic lesions were observed in many calves over 30 days of age. The importance of environmental and managerial improvement was also emphasized, since calf pneumonia tended to break out in facilities under unsatisfactory conditions in the present work.