Three cases of Pasteurella multocida infection in the respiratory tract

Pasteurella multocida (P. multocida) is well recognized as "normal flora" in the upper respiratory tract of cats, dogs and other animals. Recently, various infections due to P. multocida in human have been noted as pulmonary infections in the patients with chronic pulmonary diseases as well as skin abscesses or septicemia after an animal bite or scratch. We report here three cases of respiratory tract infections caused by P. multocida. The first two patients had acute exacerbation of bronchiectasis caused by P. multocida and the other patients with pulmonary emphysema developed pneumonia. These three patients improved by antibiotic therapy. In Japan, P. multocida respiratory tract infection is rare, but it may become more common in the future. Therefore, it seems to be important to take this pathogen into consideration in the management of chronic lung disease.     

Differentiation of Pasteurella multocida subspecies multocida isolates from the respiratory system of pigs by using polymerase chain reaction fingerprinting technique

PCR fingerprinting technique was applied to subtype 44 Pasteurella multocida subspecies multocida (P.m.sp.m.) isolates from the respiratory system of pigs. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 (core sequence of the M13 phage) grouped the 44 P.m.sp.m. strains into five distinct fingerprinting profiles, while primer 2 ((GACA)4) grouped them into seven profiles. The results suggest that PCR fingerprinting is an efficient technique to detect DNA polymorphism in the species P.m.sp.m. This technique could be used to differentiate P.m.sp.m. strains of the same capsular serotype.     

Combined enzyme and antimicrobial susceptibility profiles of caprine Pasteurella haemolytica serovar 2 respiratory tract isolates

The present investigation describes a novel method of demonstrating strain diversity among Pasteurella haemolytica biovar A, serovar 2 (PhA2) nasal turbinate isolates from a flock of 32 experimental goats during a naturally occurring outbreak of pasteurellosis. After a 21 day conditioning period in a feedyard, 51 PhA2 isolates from 27 culture-positive goats were identified including 1 on day 22, 14 on day 25, 21 on day 39, and 15 on day 66. Each PhA2 isolate was evaluated for its enzyme activity against 19 substrates with a commercial semiquantitative enzyme system and for its antimicrobial susceptibility with 12 drugs, resulting in 7 different enzyme profiles and 8 different antimicrobial susceptibility profiles. A total of 14 combined enzyme and antimicrobial susceptibility profiles were produced. The same PhA2 strain was isolated from only 4 of the 12 goats with 2 PhA2 isolations, while the same PhA2 strain was isolated from only 1 of the 6 goats with 3 PhA2 isolations. The data from this investigation demonstrated that the PhA2 upper respiratory tract flora from goats is highly heterologous.     

Effects of Pasteurella multocida toxin on porcine bone marrow cell differentiation into osteoclasts and osteoblasts

The effect of Pasteurella multocida toxin (PMT) on porcine osteoclast and osteoblast differentiation was studied using in vitro cell culture systems. When grown in the presence of Vitamin D3, isolated porcine bone marrow cells formed multinucleated cells with features characteristic of osteoclasts. Exposure of bone marrow cells to Vitamin D3 and PMT during growth resulted in formation of increased numbers and earlier appearance of osteoclasts compared to controls. Ultrafiltered medium form PMT-treated cells likewise increased osteoclast numbers, suggesting that a soluble mediator may be involved in the action of PMT. When cell cultures were treated with fluorescein-labeled PMT, fluorescence was found within the cytoplasm of small, round cells that did not resemble either osteoclasts or osteoclastic precursor cells. Cultures of porcine bone marrow cells exposed to dexamethasone, ascorbic acid, and beta-glycerophosphate developed into osteoblastic cells that formed multilayered, mineralized nodules. Exposure of osteoblastic cultures to low concentration of PMT resulted in retarded cell growth, formation of decreased numbers of nodules and minimal to no mineralization in the nodules; higher concentration of PMT resulted in increased cellular debris and poor growth of cells, with no nodule formation. These findings suggest that PMT may induce turbinate atrophy in pigs by increasing osteoclast numbers and inhibiting osteoblastic bone formation. The effect of PMT on osteoclastic differentiation and growth may not be due to a direct effect on preosteoclastic cells, but rather due to alterations in the soluble mediator secretion by marrow stromal cells.     

Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis

Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.     

Nitric oxide synthase-immunoreactive neurons in human and porcine respiratory tract

The presence of nitric oxide synthase (NO-synthase), the enzyme responsible for the production of nitric oxide (NO) from L-arginine, is shown immunocytochemically in the intrinsic neurons of the human and porcine respiratory tract. NO-synthase immunoreactivity is demonstrated in a subpopulation of neurons of the microganglia present in the wall of the extra- and intrapulmonary bronchi as well as in the hilar region of the lung in relation to blood vessels. The immunostaining was also found in some nerve fibers of the respiratory nervous system. Human and porcine lung gave similar results. The possible involvement of NO in the nonadrenergic noncholinergic (NANC) nervous regulation of the lung is discussed.     

Interaction of Mycoplasma hyopneumoniae and Pasteurella multocida infections in swine

To investigate the interaction between Mycoplasma hyopneumoniae and Pasteurella multocida infection, 32 pigs were randomly assigned by litter, sex, and weight to 4 treatment groups. Group-1 pigs were inoculated with M hyopneumoniae and allowed to recover from M hyopneumoniae infection. Group-2 pigs were vaccinated against M hyopneumoniae and then inoculated with M hyopneumoniae. Group-3 pigs were inoculated with M hyopneumoniae and developed clinical signs of mycoplasmosis. Group-4 pigs had never been exposed to M hyopneumoniae. All pigs were initially seronegative for M hyopneumoniae. All pigs were subsequently inoculated with P multocida and euthanatized 2 weeks later. Pasteurella multocida was isolated only from the lungs of group-3 pigs, and these pigs had a significantly higher median percentage of lung surface area affected by pneumonia than did pigs in the other groups. For group-3 pigs, percentage of lung surface area affected by pneumonia was positively correlated with the number of P multocida colonies isolated. We concluded that P multocida is not a primary respiratory pathogen in pigs, but that M hyopneumoniae infection can render the lungs susceptible to P multocida colonization and infection. Pigs recovered from or vaccinated against infection with M hyopneumoniae were resistant to P multocida infection.     

Resistance of bovine and porcine Pasteurella to florfenicol and other antibiotics

The resistance pattern of 221 (89 bovine, 132 porcine) pasteurella strains isolated in 1996 against 16 antibiotics or chemotherapeutics was determined by agar diffusion. Pasteurella haemolytica showed a higher level of resistance compared to Pasteurella multocida; porcine strains were more resistant than bovine strains. Over 90% of porcine Pasteurella multocida were sensible to penicillin G, ampicillin, cephalothin, polymyxin B, enrofloxacin, chloramphenicol and florfenicol. In addition, bovine strains were at least 90% sensible to oxacillin, erythromycin, gentamycin and sulfmethoxazole-trimethoprim. More than 90% of porcine Pasteurella haemolytica were classified as sensible to polymyxin B, enrofloxacin und florfenicol; bovine strains to cephalothin, neomycin und chloramphenicol as well. In 1996, 2 years after the chloramphenicol ban for food rendering animals, only 6.3% of bovine pasteurella strains proved to be resistant against chloramphenicol compared to a 16.27% fraction in 1994. The mean MIC-values of florfenicol against pasteurella spp. were nearly the same in bovine and porcine isolates with 0.53 microgram/ml and 0.52 microgram/ml respectively. Pasteurella haemolytica, however, showed higher MIC-values (0.68 microgram/ml in bovine, 0.70 microgram/ml in porcine isolates) than Pasteurella multocida with 0.47 microgram/ml in bovine and 0.51 microgram/ml in porcine strains. No isolate had a MIC of florfenicol greater than 1.0 microgram/ml, all pasteurella strains were classified sensible to florfenicol.     

Genetic analysis of antibody responses of turkeys to Newcastle disease virus and Pasteurella multocida vaccines

Heritability (h2) of 16-wk BW and primary and secondary antibody responses and genetic and phenotypic correlations among these traits were estimated for 931 male and female turkeys vaccinated with Newcastle disease virus (NDV) and Pasteurella multocida. Turkeys from a line selected for 22 or 23 generations for increased 16-wk BW were vaccinated at 6 and 12 wk of age with blood samples collected 3 wk postvaccination. Antibody titers were determined using an ELISA method and transformed to log(e) for analysis. Heritability estimates for primary and secondary antibody responses to NDV were .380 +/- .070 (SE) and .296 +/- .063, respectively. For primary and secondary antibody responses to P. multocida, h2 estimates were .458 +/- .075 and .333 +/- .066, respectively. Heritability estimate for 16-wk BW was .404 +/- .071. The genetic correlation between primary and secondary antibody responses to NDV was .491 +/- .150. There was no genetic correlation between primary and secondary antibody responses to P. multocida. Although the genetic correlation between primary antibody responses to NDV and P. multocida was .292 +/- .159, the genetic correlation between secondary responses to the two antigens did not differ from zero. There were no genetic correlations between antibody responses and 16-wk BW. Similar results were observed for phenotypic correlations. Based on heritability and genetic correlation estimates, it would be possible to improve antibody responses to either NDV or P. multocida singularly; however, to improve antibody responses to both antigens, selection would have to be applied for each antigen.     

Antibody responses of cattle to outer membrane proteins of Pasteurella multocida A:3

OBJECTIVE: To quantify the serum antibody responses to Pasteurella multocida A:3 outer membrane proteins (OMP) for cattle vaccinated with the homologous serogroup and to correlate those responses with the extent of experimentally induced pneumonia. ANIMALS: 29, 5- to 8-month-old beef-type calves. PROCEDURE: Calves were vaccinated SC or by aerosal exposure on days 0 and 7 with live or killed P multocida or phosphate-buffered saline solution (control) and subsequently challenge exposed with virulent P multocida. Antibody responses to P multocida A:3 outer membranes were quantified, using an ELISA. Antibody responses to individual OMP were detected by immunoblot analysis (western blot) and were quantified by densitometry. Antibody responses were compared among groups of calves and for various times after vaccination. Regression analyses were used to determine whether significant correlations existed between lesion scores and antibody responses to either whole outer membranes or to individual OMP. RESULTS: By ELISA, antibody responses to outer membranes for calves aerosol vaccinated with live P multocida were significantly (P < 0.05) greater than those for control calves or for killed P multocida vaccinates. There was a significant (P < 0.05) correlation between lesion score and antibody responses to outer membranes. By western blotting and densitometry, antibodies to 11 prominent OMP (100, 97, 90, 85, 74, 53, 46, 35, 32, 21, and 16 kd) were identified and quantified. In experiment 1, SC vaccination with live P multocida increased antibody binding to all protein bands except 85-, 74-, and 35-kd bands. Aerosol vaccination with live P multocida stimulated increases in antibody binding to all bands except 100 and 16 kd. Antibody responses to the 97-, 90-, 74-, and 35- kd bands were significantly (P < 0.05; greater for live aerosol vaccinates than for control calves. In experiment 2, antibody responses were not different between the killed P multocida vaccinates or control calves Antibody responses for live P multocida aerosol vaccinates were significantly (P < 0.05) greater than those for control calves for the 100-, 90-, 85-, 74-, 53-, 35-, and 16-kd bands. Regression analyses indicated significant correlations (P < 0.05) between lesion score and antibody responses to the 100-, 90-, 53-, 46-, 35-, and 32-kd OMP. CONCLUSIONS: Several OMP of P multocida type A:3 may be important for stimulating immunity to the organism in cattle.     

Localization of the intracellular activity domain of Pasteurella multocida toxin to the N terminus

We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqalpha protein that is coupled to phosphatidylinositol-specific phospholipase Cbeta1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268-1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.     

Proteins found within porcine respiratory tract secretions bind lipopolysaccharides of Actinobacillus pleuropneumoniae

Affinity for porcine respiratory tract secretions was found in some isolates of Actinobacillus pleuropneumoniae and involved lipopolysaccharides (LPS) (M. Bélanger, S. Rioux, B. Foiry, and M. Jacques, FEMS Microbiol. Lett. 97:119-126, 1992). In the present study, the affinity for a crude preparation of porcine respiratory tract mucus of isolates of the Pasteurellaceae family, i.e., Actinobacillus, Haemophilus, and Pasteurella spp., and of some unrelated gram-negative bacteria was examined. Affinity for crude porcine respiratory tract mucus was not a property shared by all Pasteurellaceae isolates tested. Furthermore, affinity for the porcine crude mucus preparation was not unique to the Pasteurellaceae group and did not seem to be restricted to bacteria originating from pigs. Different surface properties of A. pleuropneumoniae isolates in relation to their adherence to crude mucus were examined. The capsular layer seemed to mask the adhesin and interfered with adherence to crude mucus. Two poorly capsulated isolates, which had a more hydrophobic surface and bound Congo red, were also heavily labeled by gold particles coated with polymyxin, which is known to interact with the lipid A-core region of LPS, and adhered strongly to respiratory tract secretions. Tetramethylurea, charged polymers, divalent cations, chelators, monosaccharides and amino sugars, or lectins were unable to inhibit adherence of A. pleuropneumoniae to the crude mucus preparation. To identify the receptor(s) recognized by the lipopolysaccharidic adhesin of A. pleuropneumoniae, affinity chromatography was used. Two bands, which were proteinaceous in nature, of 10 and 11 kDa were recovered. Our results suggest that two low-molecular-mass proteins present in porcine respiratory tract secretions bind A. pleuropneumoniae LPS.     

Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida

Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. We utilized transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild-type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of truncated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA polymerization. This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected activity in membrane preparations in vitro. The polypeptide size was verified by photoaffinity labeling of the native P. multocida HA synthase with azido-UDP sugar analogs. Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV-1 virus, or vertebrates. Instead, a portion of the central region of the new enzyme is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. In summary, we have discovered a unique HA synthase that differs in sequence and predicted topology from the other known enzymes.     

Physical and genetic map of the Pasteurella multocida A:1 chromosome

A physical and genetic map of the Pasteurella multocida A:1 genome was generated by using the restriction enzymes ApaI, CeuI, and NotI. The positions of 23 restriction sites and 32 genes, including 5 rrn operons, were localized on the 2.35-Mbp single circular chromosome. This report presents the first genetic and physical map for this genus.     

Comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure

OBJECTIVE: To compare the virulence of selected strains of porcine reproductive and respiratory syndrome virus (PRRSV) relative to reproductive performance of pregnant gilts. DESIGN: 16 pregnant gilts (principals) were exposed oronasally to 4 strains (vaccine strain RespPRRS, field strains VR-2385, VR-2431, and NADC-8, 4 gilts/strain) of PRRSV on or about day 90 of gestation, 4 pregnant gilts (controls) were kept under similar conditions, except for exposure to PRRSV. Samples and specimens obtained from gilts, pigs (before ingestion of colostrum), and fetuses were tested for PRRSV and homologous antibody. ANIMALS: 20 pregnant gilts. PROCEDURE: The virulence of each strain of PRRSV was evaluated mainly on the clinical status of the corresponding litters at farrowing. RESULTS: Most gilts remained clinically normal throughout the study and farrowed normally at or near the expected farrowing time. All virus strains crossed the placenta of principal gilts to infect fetuses in utero. The number of late-term dead fetuses (which appeared to be the best measure of relative virulence) ranged from 0 for litters of control gilts and gilts exposed to strain RespPRRS, to 38 for gilts exposed to strain NADC-8. All principal gilts became viremic and developed antibody against PRRSV. All strains persisted in alveolar macrophages of at least some principal gilts for at least 7 weeks after exposure. CONCLUSION: Strains of PRRSV vary in virulence. CLINICAL RELEVANCE: The effects of PRRSV on reproductive performance are strain dependent and this should be considered in making a tentative diagnosis on the basis of clinical observations.     

Isolation of porcine respiratory coronavirus from pigs affected with porcine reproductive and respiratory syndrome

Four cytopathogenic viruses were isolated in CPK cells derived from porcine kidneys from tonsils and lungs of 3 of 15 pigs affected with porcine reproductive and respiratory syndrome virus. Physicochemically and morphologically, the isolates were similar to a coronavirus. The isolates were not distinguished from transmissible gastroenteritis virus (TGEV) by a neutralization test using polyclonal antibodies, but differentiated from TGEV by monoclonal antibodies capable of discriminating between TGEV and porcine respiratory coronavirus (PRCV), indicating that the isolates were PRCV. In a serological survey of 30 serum samples each collected from about 50 days old pigs in the 2 affected farms, 29 (97%) and 15 (50%) sera were positive for neutralizing antibody against the isolate with the titers ranging from 2 to 64, respectively.     

Two closely related adenovirus genome types with kidney or respiratory tract tropism differ in their binding to epithelial cells of various origins

The host cell interactions of the genome types Ad11p and Ad11a of human adenovirus serotype 11, displaying kidney or respiratory tropism, were compared using FACS analysis. Kinetic experiments indicated that the virus binding stated immediately and reached a plateau after 30 min. The binding of biotinylated virions to seven continuous cell lines. A549, A498, J82, HeLa, CHO, MDCK, and human diploid fibroblasts (HEDF), was quantitated by FACS analysis. The binding capacities of the two viruses to all human cell lines but A549 cells appeared to differ. Ad11p virions manifested high affinities, whereas Ad11a virions presented low affinities. Neither of the two viruses bound to CHO or MDCK cells. Reciprocal competition experiments showed that the Ad11a virions could be weakly blocked by the Ad11p virions, whereas the Ad11p virions could not be competed at all by the Ad11a virions. The binding of the Ad11p virions to cells could be blocked by the rfiber antiserum of Ad11p, but not by the corresponding antiserum against Ad11a or Ad35p. A comparison of the cytopathogenicity of the seven cell lines infected by Ad11p and Ad11a demonstrated that the efficiency of the initial event of an adenovirus infection directly affects the outcome of the viral infection. The Ad11a in the A498, J82, HeLa, or HEDF cells that presented lower affinity and receptor concentration showed 100 times less infectivity than that in A549 cells displaying high affinity and receptor concentration. These results indicate that the cell susceptibility to Ad11p and Ad11a infection strongly depends on both the number of fiber receptors on the host cells and the receptor affinity for ligands on the fiber knob. Our findings also suggest that the receptors for Ad11p and Ad11a on the surface of different cell types may be different or on different sites.