Resistance of some capsular serotype D strains of Pasteurella multocida to rabbit polymorphonuclear neutrophil phagocytosis

The mechanism of resistance of Capsular Type D strains of Pasteurella multocida to killing by rabbit polymorphonuclear neutrophils (PMN) was studied using an in vitro assay that differentiates intra- from extracellular bacteria. Two Capsular Type D strains (3761 and 3766), resistant to killing by rabbit PMN, and one Type A strain (R1), susceptible to PMN destruction, were compared. After combining opsonized bacteria and PMN, the Capsular Type D Strains 3761 and 3766 remained extracellular while the Capsular Type A Strain R1 was internalized by PMN. Thus, both Type D strains were resistant to phagocytosis by rabbit PMN.     

A method to detect rabbit neutrophil phagocytosis of Pasteurella multocida

A method was developed to detect neutrophil phagocytosis of bacteria by determining whether neutrophil-associated bacteria were intra- or extracellular. Neutrophils were treated with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride to inhibit degranulation and, consequently, killing of bacteria. Treated neutrophils and opsonized Pasteurella multocida were combined. Following phagocytosis, the suspensions were centrifuged and the pellets were washed to remove non-cell-associated bacteria. The pellets were resuspended and heparin was added to prevent further phagocytosis. Samples were removed, and the number of viable bacteria was determined by a dilution and plate count technique. Streptomycin, an antibiotic that is poorly taken up by neutrophils, was added to kill extracellular bacteria, and the suspensions were incubated for 20 minutes at 37 C, and samples were removed again and bacterial numbers were determined. Percentage killing of bacteria by streptomycin was calculated. Phagocytosed bacteria were protected from the bactericidal action of streptomycin.     

A new serotype of Pasteurella multocida associated with fowl cholera

Gel-diffusion precipitin tests demonstrated an additional Pasteurella multocida serotype, designated serotype 16. Isolate P-2723, antigenically distinct from the other (previously reported) 15 serotypes, was from a turkey affected with fowl cholera. This serotype is not widely distributed. Isolate P-2723 was of mild virulence in turkeys, resulting in local infections in the hock joint and sternal bursa of only 1 of 9 turkeys exposed.     

Effects of the lipopolysaccharide-protein complex and crude capsular antigens of Pasteurella multocida serotype A on antibody responses and delayed type hypersensitivity responses in the chicken.

The effects of the lipopolysaccharide-protein complex (LPS) and crude capsular antigen (CCA) prepared from Pasteurella multocida serotype A isolated from a duck in the Philippines, on antibody responses to sheep red blood cells (SRBC) and Brucella abortus (BA) and delayed type hypersensitivity (DTH) responses to bovine serum albumin (BSA) in the chickens were studied. Chickens injected subcutaneously with LPS and CCA at 1 and 2 weeks of age and immunized intravenously with the mixed antigens of SRBC and BA, at 3 and 4 weeks of age showed significantly increased antibody responses against both SRBC and BA, when evaluated at 7 days after each immunization. In addition, these chickens sensitized intramuscularly with the emulsion of BSA in complete Freund's adjuvant at 5 weeks of age, and then injected into the wattle with BSA at 7 weeks of age also showed significantly increased DTH responses against BSA, when evaluated at 24 and 48 hr after challenge. These results indicate that LPS and CCA of P. multocida serotype A have a property enhancing humoral and cell-mediated immune responses.     

Bovine pneumonic pasteurellosis: chemiluminescent response of bovine peripheral blood leukocytes to living and killed Pasteurella haemolytica, Pasteurella multocida, and Escherichia coli

A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phagocytic uptake and killing of virulent and avirulent strains of Pasteurella multocida of capsular serotype A by chicken macrophages

The susceptibility of two highly virulent (VP161 and VP138) and two less virulent (VP17 and VP21) strains of Pasteurella multocida to phagocytic uptake and killing by chicken macrophages was compared using in vitro phagocytosis and bactericidal assays. When compared with VP17 and VP21, particularly after they were preopsonised with specific immune serum, VP161 and VP138 were more resistant to phagocytosis by chicken macrophages. The uptake of these bacteria increased following the removal of the bacterial capsules with hyaluronidase. All strains preopsonised with specific immune serum were killed to some extent by chicken macrophages. However, the percentages of killing for VP17 and VP21 were higher than those of VP161 and VP138. When the capsules of VP161 and VP138 were removed, the susceptibility of the bacteria to bactericidal activity of chicken macrophages increased. It can be concluded that the virulent strains of P. multocida were more resistant to phagocytosis and phagocytic killing by chicken macrophages compared with the less virulent strains. The hyaluronic acid capsule was considered to be important in the resistance, but might not be the only factor contributing to the resistance since the less virulent strains of P. multocida also possess capsules.     

Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera.

The protein profiles of Pasteurella multocida serotype 1 isolates and the response of chickens to serotype 1 antigens were investigated using SDS-PAGE. Patterns obtained with Coomassie blue staining of soluble protein extracts were similar. The major difference between isolates was the position of one of the major proteins in the 34-38 kDa region. When chickens were experimentally infected with a clinical isolate of P. multocida serotype 1 various proteins were recognised by immunoblotting, including one with a relative molecular weight of 34 kDa; however, no reactions were observed in the region where LPS is known to migrate. When these infection sera were used in an EIA with purified LPS obtained from Heddleston serotype 1 type strain (X-73) they reacted strongly. Serum used for serotyping isolates in the gel diffusion precipitin test recognised many antigens in common with sera from infected birds, but some antigens were specific to typing sera.     

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 x 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.     

Characterization of toxin from different strains of Pasteurella multocida serotype A and D

Chromosomal DNA from 13 different selected Pasteurella multocida spp. multocida strains of serotypes A and D were isolated and compared. All 10 toxigenic strains were recognized by a DNA probe which included the toxA gene coding for the Pasteurella multocida toxin (PMT). None of the three nontoxigenic strains reacted with the DNA probe. Toxin from the 10 toxigenic strains were isolated and compared. All were found to possess the biological characteristics previously described for the PMT isolated from P. multocida ssp. multocida NCTC 12178, including molecular mass of approx. 143 kDa and reactivity with a series of monoclonal antibodies. Toxin prepared from different toxigenic strains could not be differentiated immunologically by tandem crossed immunoelectrophoresis, Toxin, which was affinity purified from four of the strains and subsequently inactivated by formaldehyde, was cross-protective when used for vaccination of mice before challenge with PMT. It is concluded that the toxin from toxigenic strains of P. multocida ssp. multocida must be very similar, if not identical.     

Resistance of Pasteurella multocida A:3,4 to phagocytosis by turkey macrophages and heterophils

A virulent field isolate and 2 vaccine strains of Pasteurella multocida A:3,4 were compared for resistance to phagocytosis by turkey macrophages and heterophils, using in vitro assays. The least virulent vaccine strain (M-9) was phagocytosed to a greater degree than was the field isolate or the other vaccine strain (Clemson University). All 3 bacteria differed significantly from each other in the amount of capsular material present as measured by polycationic ferritin labeling and electron microscopy. Removal of the capsule with hyaluronidase resulted in a significant increase in phagocytosis of the field isolate.     

Survival of avian strains of Pasteurella multocida in chicken serum

The ability of bacteria to survive in serum is considered a likely virulence determinant in diseases where the infective bacteria become septicaemic. Optimal conditions were established to test the survival of Pasteurella multocida in chicken serum. Serum was used at 90%, the inoculum was 10(3)-10(4)cfu in phosphate buffered saline pH 7.4. Survival was measured after incubation for 2-4 h; if survival was <50% the strain was considered serum susceptible. Susceptible strains were either killed or their growth was inhibited. Some resistant strains not only survived but grew rapidly in unheated serum. Thirty-five strains, all originally isolated from clinical fowl cholera, were tested; eight were susceptible, of which three were killed and five inhibited, and the remainder (27) were resistant. Ten serum-resistant P. multocida serogroup A strains were grown in hyaluronidase to remove the capsule and survival in chicken serum was re-tested. Three strains became susceptible, while seven strains remained resistant. Three serum susceptible strains were then tested in the presence of cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA). This substance is present in the human serum, and is known to mask the effect of complement on Neisseria gonorrhoeae rendering susceptible strains resistant. Two of the three serum susceptible strains became resistant in the presence of CMP-NANA. Serum susceptibility/resistance was more complex than that of Escherichia coli, and the role of resistance to avian complement in the pathogenesis of fowl cholera remains to be determined.     

Effects of Pasteurella multocida endotoxins on turkey poults

Studies of turkey poult responses to Pasteurella multocida endotoxins indicated that lipopolysaccharide (LPS) preparations from two highly pathogenic strains and free endotoxin from one of these strains were all similar in lethal toxicity. Lethal intravenous doses were generally high, 1 mg or more for 1-week-old poults (13.3 mg/kg). The toxic effects of LPS were not increased by repeated administration of small hourly doses. For both forms of endotoxin, the relationship between dose and response was considered erratic. Attempts to increase the susceptibility of poults to LPS by administering a liver-damaging substance (galactosamine) or a histamine-releasing substance (compound 48/80) or by performing surgical bursectomy were not effective. The LPS did not provoke a dermal Shwartzman reaction, even though doses used were 10 times those that produced a characteristic reaction in a rabbit.     

Fowl cholera epornitic: antigenic characterization and virulence of selected Pasteurella multocida isolates.

An epornitic of fowl cholera involving turkey flocks of several farms within a 15-mile radius in Utah was studied. Pasteurella multocida strains isolated from birds in affected flocks were antigenically characterized as A:1, A:3, and B:4, based on capsular sero-grouping and somatic serotyping results. Experimental exposure of poults with each of two strains representing the rarely reported capsular group B indicated that both were virulent.     

Septicemia in vaccinated and nonvaccinated turkeys inoculated with Pasteurella multocida serotype A:3,4

Sixty-four, 10-week-old turkeys were inoculated with a highly virulent field isolate (86-1913) of Pasteurella multocida serotype A:3,4 by an oculo-nasal-oral route. Inoculated turkeys were examined at 4, 8, 16, 20, and 24 hours post-inoculation for bacteremia and histologic lesions. Bacteremia was detected in one of six turkeys 8 hours after inoculation and in four of six turkey poults at 16 hours post-inoculation. Pasteurella multocida was isolated from the spleens of two turkeys at 8 hours and from the spleens of all six poults 16 hours after inoculation. Peak concentrations of P. multocida reached 10(9) colony forming units per ml of blood. At 4 to 8 hours post-inoculation, isolate 86-1913 produced a fibrinopurulent bronchopneumonia followed by severe pulmonary necrosis, pleuritis, vasculitis; and, at 16 to 24 hours post-inoculation numerous extracellular bacteria were observed. Hepatic lesions included focal heterophil aggregates 8 hours after inoculation; these progressed to hepatic necrosis. Numerous extracellular bacteria within sinusoids were present 16 to 24 hours after inoculation. At 16 to 24 hours post-inoculation, there was degeneration of periarteriolar reticular cells in the spleen; these cells progressed to coalescing coagulative splenic necrosis with extracellular bacterial colonies. A second group of 41, 10-week-old turkeys, previously vaccinated with the Clemson University strain of P. multocida serotype A:3,4, were challenged with isolate 86-1913.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of outer membrane proteins participating in iron transport in Pasteurella multocida serotype A3

Iron-regulated outer membrane proteins (IROMPs) of P. multocida serotype A3, which function as receptors for complexes containing iron ions, are induced by iron deficiency in the bacterial growth environment. Analysis of an electrophoresis image of proteins isolated from bacteria grown on medium supplemented with 2,2'-dipyridyl revealed expression of 16 new proteins that were not noted in the case of the bacteria grown in standard conditions, with molecular weights from 30 to 160 kDa. Induction of IROMP expression occurred within 30 minutes after restricted iron conditions were established. In immunoblotting, distinct reactions were noted for proteins of molecular weight ranges of 25-49 kDa, 61-95 kDa, and 108-214 kDa. Proteins of the molecular weight of 68, 75 and 86 kDa were analysed using mass spectrometry and matched with the highest probability to proteins in the NCBI data base. Several dozen different proteins with similar amino acid sequences were matched to each sample.     

Cytokine profiles,apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P < 0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.