DNA base composition of rickettsiae.

There is a small but distinct difference in DNA base composition between the typhus and spotted fever groups of rickettsiae. The molar percentages of guanine plus cytosine for Rickettsia prowazeki, R. typhi, and R. canada are approximately 30, for R. rickettsi, R. conori, and R. akari they are about 32.5. The percentage for trench fever rickettsia, Rochalimaea quintana, is 38.6.

Outer membrane proteins of bovine strains of Pasteurella multocida type A and their doubtful role as protective antigens.

Outer membranes were prepared by the Sarkosyl method from 30 strains of Pasteurella multocida and the closely related Taxon 13, which had been isolated from cattle. The patterns of the outer membrane proteins (OMPs) on SDS-PAGE were generally similar to one another, though the four major proteins (a-d) varied somewhat in molecular mass; these patterns allowed the strains to be arranged into 12 groups. Taxon 13 strains and typical P. multocida strains were indistinguishable, both types being found within the same group. Mice were vaccinated with heat-killed bacteria of three strains and challenged with 10 LD50 of homologous and heterologous live bacteria, representing groups based on OMP patterns; the best protection was afforded by strain W674, which protected against nine of the 17 challenge strains; but there was no correlation between protection and PAGE pattern. Pre-vaccination and pre-challenge sera were used in immunoblotting to probe OMPs from protective and non-protective strains. All three vaccines produced antibody to proteins a and d; these proteins appeared to be common to all strains, varying in molecular mass but not in overall antigenic expression. The antibody response to the other two major OMPs appeared to be PAGE-group specific. There was no correlation between protection and the antigen pattern seen by immunoblotting.

Cytotoxin from an ovine strain of Pasteurella haemolytica: characterisation studies and partial purification.

A cell-free, water-soluble cytotoxin from an ovine strain of Pasteurella haemolytica biotype A serotype 1 killed sheep bronchoalveolar macrophages at 37 degrees C, but not at 4 degrees C or 22 degrees C. The cytotoxin was stable over the pH range 2-12, resistant to heat at 60 degrees C but inactivated at 100 degrees C or by autoclaving. Trypsin also destroyed the cytotoxin, which is therefore thought to contain a protein component essential for biological activity. A preliminary purification of the crude cytotoxin using gel-filtration column chromatography resulted in the isolation of a biologically active fraction which resolved as a single protein band and one carbohydrate band on non-dissociating polyacrylamide gels. However, this fraction resolved into approximately 16 component bands on a sodium dodecyl sulphate polyacrylamide gel.

A colourimetric, microplate assay for the leucotoxin of Pasteurella haemolytica.

Culture supernates of Pasteurella haemolytica, which contain leucotoxin, inhibited the reduction of nitroblue tetrazolium (NBT) by bovine and ovine but not rabbit leucocytes in response to phorbol 12-myristate 13-acetate (PMA). Culture supernates of P. multocida, which contain no leucotoxin, had no inhibitory effect on the response of leucocytes from any species. The inhibition of NBT reduction was assessed visually or spectrophotometrically in the wells of microplates and used as a simple assay for leucotoxin. It was as sensitive as the trypan blue dye-exclusion method and did not require the use of radioisotopes. In addition, sera from P. haemolytica-infected calves inhibited leucotoxin activity in the microplate assay. Thus, inhibition of NBT reduction after stimulation of ruminant leucocytes with PMA can be used as a simple, specific assay for leucotoxin and for anti-leucotoxin antibodies.

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Taxonomy of pasteurella anatipestifer. 2. Cellular fatty-acid profile by gas chromatography.

Methylated cellular fatty acids of representative strains of Pasteurella spp., Moraxella spp., and P. anatipestifer were subjected to gas chromatography in an attempt to further support the independence of P. anatipestifer from both Pasteurella and Moraxella. All Pasteurella spp. and Moraxella spp. revealed group characteristics specific for each genus that could be easily differentiated from the unique profile of P. anatipestifer. All P. anatipestifer strains tested showed similar fatty-acid profiles in gas chromatography, regardless of host of origin.

Studies of Pasteurella anatipestifer: an approach to its classification.

Moraxella spp., Pasteurella spp., and strains of P. anatipestifer were tested for biochemical reactions, growth temperature, viability, antibacterial sensitivity, and DNA base composition. P. anatipestifer was viable for shorter periods at 37 C, showed high resistance to polymyxin B and kanamycin, and had lower base composition than reported for Moraxella and Pasteurella spp. Because of these conditions, P. anatipestifer should be excluded from the genera Pasteurella and Moraxella.

Pasteurella anatipestifer infections in California turkey flocks: circumstantial evidence of a mosquito vector.

Outbreaks of Pasteurella anatipestifer infections in California turkey flocks were investigated and found to have a seasonal distribution, with a peak incidence in fall, coinciding with peak Culex mosquito populations. An experiment was conducted to test the hypothesis that mosquitoes may serve as vectors for P. anatipestifer infections in turkeys. Four 7-week-old turkey poults were exposed for 7 days to mosquitoes captured from turkey barns during a field outbreak of P. anatipestifer serotype 1 infection. One turkey developed serum antibodies to serotype 1, detectable by enzyme-linked immunosorbant assay, and was resistant to an intravenous inoculation of P. anatipestifer serotype 1 at 4 weeks postexposure. Giemsa-stained blood smears from this bird and from three 7-week-old turkeys inoculated intravenously with P. anatipestifer revealed the presence of rod-shaped bacteria in or on the surface of host erythrocytes. No such rod-shaped bodies were found on erythrocytes of an uninoculated control turkey.

Protein electrophoretic pattern of Pasteurella haemolytica.

Electrophoresis in polyacrylamide gels of the constituent proteins of the 12 serotypes and an untypable strain of Pasteurella haemolytica showed a pattern of bands that divided the group into two. This division conformed to the A and T biotype groupings of Smith (1959) although the serotype A9 showed only minor band difference from the three T serotypes 3, 4 and 10. It was not possible by this method to separate all the type strains from each other by the specific recognition of the patterns of protein mobilities produced.

Presence of bacterial glycocalyx and fimbriae on Pasteurella haemolytica in feedlot cattle with pneumonic pasteurellosis.

This investigation was conducted to determine if Pasteurella haemolytica within feedlot cattle affected by pneumonic pasteurellosis express fimbriae (pili) and bacterial glycocalyx. Bacteriological culture of pulmonary tissue from three calves with fibrinous pneumonia resulted in heavy growth of P. haemolytica. Transmission electron microscopy of the lungs showed numerous microcolonies of gram-negative bacteria with morphology typical of Pasteurella haemolytica. The cells within these microcolonies possessed bacterial glycocalyces which stained with ruthenium red. Glycocalyx-encased microcolonies were also present in specimens examined by scanning electron microscopy. Typical P. haemolytica cells were evident in a tracheal specimen and these bacteria had radial glycocalyces consistent with polysaccharide and proteinaceous material condensed on linear structures suggestive of fimbriae. The pathogenetic importance of the bacterial glycocalyx and fimbriae in shipping fever pneumonia has yet to be established but their presence in clinical cases of Pasteurella pneumonia in feedlot cattle further supports a possible role in the initiation and progression of this disease as well as bacterial resistance to antimicrobial agents.

Electron microscopic description of glycocalyx and fimbriae on the surface of Pasteurella haemolytica-A1.

Several electron microscopic techniques were used to examine the surface of cells of Pasteurella haemolytica (biotype A, serotype 1) grown in vitro. All methods showed the presence of a very extensive glycocalyx on logarithmic phase (6 h) cells grown in liquid media. The anionic glycocalyx of these cells stained well with ruthenium red, but collapsed during dehydration for electron microscopy unless stabilized with specific antibodies. When the same techniques were used to examine cells in the stationary phase (18 h) the glycocalyx was much reduced. Large numbers of fimbriae were seen on both 6 h and 18 h cells grown in fluid media without shaking. In summary, logarithmic phase cells of P. haemolytica have both fimbriae and extensive anionic glycocalyx at their surface and we suggest that either or both of these structures may be important in the colonization of the bovine respiratory tract and the subsequent pathogenesis of Pasteurella pneumonia.

Electron microscopic examination of cells of Pasteurella haemolytica-A1 in experimentally infected cattle.

Several modern electron microscopy techniques were used to examine Pasteurella haemolytica (biotype A, serotype 1) (strain B122) recovered from experimentally infected cattle and in situ within the lung tissue of experimentally infected cattle. Glycocalyx four to five times thicker than that seen on P. haemolytica grown in vitro was evident on bacterial cells recovered from live infected calves by pulmonary lavage. Fimbriae were also present on cells recovered by lavage. A thick glycocalyx was also seen on P. haemolytica-A1 within the lungs of experimentally infected cattle at necropsy. In summary, cells of P. haemolytica-A1 in experimentally infected cattle have fimbriae and glycocalyx on their cell surfaces and these structures appear to be important in bacterial colonization of the bovine respiratory tract and pathogenesis of shipping fever (Pasteurella) pneumonia.

Characterization of outer membrane protein-enriched extracts from Pasteurella multocida isolated from turkeys.

Outer membrane protein (OMP)-enriched extracts of avian strains of Pasteurella multocida were examined by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium did not have a significant effect on the OMP profiles of strains of P multocida examined; however, in vivo propagation had an appreciable effect on the OMP profile composition of the reference strain P-1059. Such bacteria, expressed several additional OMP in the 27-kD, 48-kD, 56-kD, 60-kD, 80-kD, and 94-kD molecular mass regions. These OMP were not detected in the electrophorogram of strain P-1059 grown in vitro. The OMP profiles of reference strains of the 16 serotypes of P multocida did not identify any serotype-specific protein markers. Field strains of serotype A:3 had variation in OMP profiles and did not express OMP that all were identical to that expressed by the reference strain P-1059. The live attenuated CU and M9 bacterial vaccine strains expressed strain-specific OMP markers of 48-kD and 45-kD molecular masses, respectively. These strain-specific OMP markers may be used to differentiate these strains from virulent field strains that are of the same serotype and isolated from turkeys that have succumbed to pasteurellosis as a result of vaccine-related reactions or breakdown in immunity.

Purification and partial characterization of a macrophage cytotoxin from Pasteurella haemolytica.

A protein from Pasteurella haemolytica that was highly immunogenic and toxic toward bovine alveolar macrophages was partially purified. When isolated from culture supernatants of P haemolytica serotype 1 or serotype 6, the protein reacted on Ouchterlony immunodiffusion tests with antisera from 12 serotypes of P haemolytica, but did not cross-react with antisera to serotypes of P multocida. This indicated that the protein may be specific for P haemolytica. Bacteria were grown in dialysis culture in a brain-heart infusion and calf-serum growth medium. The protein was isolated from the medium by ultrafiltration and size-exclusion chromatography and has a molecular weight of approximately 150,000 daltons. The protein, which is highly immunogenic and has the characteristics of a virulence factor, is common to all serotypes of P haemolytica, and may be an effective agent for immunization against P haemolytica in cattle.

Extraction of capsular material from Pasteurella haemolytica.

Water and phosphate-buffered saline solution suspensions of early log-arithmic phase cells of Pasteurella haemolytica were incubated for 1 hour at various temperatures to remove capsular material with a minimum of cell lysis or death. Criteria used to determine capsular removal included change in agglutinability of the organism and disappearance of an antigenic component by the fluorescent antibody test and the agar-gel diffusion technique. The capsular material could be removed in a saline solution suspension at 41 C with little decrease in viability, thereby providing comparable cell populations with and without capsules for use in subsequent studies.

Failure of ribosomes from nonencapsulated Pasteurella multocida to protect CF-1 mice.

Monomeric ribosomes (70S) were isolated from nonencapsulated Pasteurella multocida strain X-73 by mechanical disruption and by chemical lysis. Contamination of the initial preparations by lipopolysaccharide (LPS) was evident by gel immunodiffusion. Gel filtration chromatography of the 70S ribosomes resulted in ribosomes with LPS contamination below the concentration that was detected by gel immunodiffusion, but chickens vaccinated with these preparations responded serologically to LPS by producing a detectable titer of passive hemagglutination antibody. Washing the 70S ribosomes through 0.5 M NH4Cl and 30% sucrose rendered them virtually free of contamination by LPS as evidenced by lack of passive hemagglutination titer. Ribosomes were evaluated as protective immunogens in CF-1 mice. Two inoculations (14 days apart) at each dosage level of 0.4, 40.0, and 400 microgram/mouse were given. At 21 days after vaccination, the mice were challenge exposed with encapsulated P multocida X-73 cells. None of the ribosomal preparations (crude or purified) protected against challenge exposure. Killed whole nonencapsulated cells did protect against challenge exposure. In contrast to numerous reports on the effectiveness of ribosomes as protective immunogens, ribosomes from nonencapsulated P multocida cells were not protective immunogens in mice challenge exposed with encapsulated cells under these conditions.

Plasmid profile analysis of bovine isolates of Pasteurella haemolytica.

Pasteurella haemolytica isolates, obtained from cattle with respiratory tract disease, were characterized as to serotype, antimicrobial susceptibility, and plasmid content. Strains isolated from 2 groups of cattle were compared. Remarkable similarity was seen in the plasmid profiles of isolates of the same serotype. In contrast, isolates of 2 different serotypes had totally different plasmid profiles.

Resistance plasmids of Pasteurella multocida isolated from turkeys.

From 1940 through 1978, fifty-eight strains of Pasteurella multocida (serotype 3) were isolated from turkeys throughout the United States and were examined for R-plasmids. Forty-one of the isolates contained plasmid DNA, of which 7 isolates were found to encode resistance to tetracycline, streptomycin, and sulfonamides, or to streptomycin and sulfonamides. The R-plasmids were 2 to 10 megadaltons, nonconjugal, and contained a moles percent guanine plus cytosine ratio in the range of 57 to 61. The R-plasmids did not belong to any of the 19 incompatibility groups evaluated, including Inc Q. Digestion with restriction endonuclease indicated that 2 of the plasmids from P multocida isolated in 1960 and 1962 were identical, whereas 4 of the 5 plasmids obtained from P multocida isolated after 1966 were identical, with the 5th plasmid closely related to the other 4. The results indicated that R-plasmids were not widely dispersed among P multocida (serotype 3) isolated from turkeys in the United States. The nontransmissible nature of these plasmids was probably the major reason for their lack of dissemination.

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica.

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.

Properties of free endotoxin from Pasteurella multocida.

Free endotoxin (FET) from virulent encapsulated Pasteurella multocida or from an avirulent nonencapsulated mutant is capable of inducing active immunity, but the lipopolysaccharide (LPS) moiety of the endotoxin is not. These results suggest that a protein of P multocida is involved in the stimulation of active immunity. The serologic specificity of the FET is associated with the LPS moiety, which is related to a heat extracted antigen that is used for serotyping P multocida. The FET is capable of producing widespread vascular alteration and death. It is present in the vascular system of turkeys with acute fowl cholera, and it can be detected with the "Limulus" test for endotoxins and with the gel diffusion precipitin test.