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Modulation of the humoral immune response of swine and mice mediated by toxigenic Pasteurella multocida.

Progressive atrophic rhinitis is an upper respiratory tract disease of pigs caused by toxigenic strains of the bacterium Pasteurella multocida. In this study the effect of P. multocida on the humoral immune response of pigs and mice was investigated. Pigs were given live intranasal challenge with either a toxigenic strain or a non-toxigenic strain of P. multocida, or were given daily intranasal instillation of a cell-free lysate of the toxigenic strain. Mice were given a live intranasal challenge of either a toxigenic or a non-toxigenic strain of P. multocida. All of the animals were immunised with ovalbumin and serum concentrations of anti-ovalbumin antibodies were quantified and compared between different treatment groups and control animals. Intranasal challenge with toxigenic P. multocida caused a significant reduction in the levels of anti-ovalbumin IgG in both species. A similar effect was seen in pigs given a cell-free extract of toxigenic P. multocida. Whilst the mechanism of this suppression is unclear, we surmise that immunomodulation of the host is an important virulence factor for toxigenic P. multocida, and could be an important function of the toxin. This immunomodulatory effect may enhance colonisation of P. multocida aiding horizontal transmission and may predispose to concurrent infection with other potential pathogens.

Animals↗

In vitro susceptibility of Pasteurella multocida subspecies multocida strains isolated from swine to 42 antimicrobial agents.

The minimal inhibitory concentrations (MICs) of 42 antimicrobial agents were determined against 59 strains of Pasteurella multocida subspecies multocida, all isolated from swine lungs with lesions indicative of pneumonia. Penicillins (except cloxacillin), aminoglycosides, tetracyclines, erythromycin, josamycin, thiamphenicol, colistin, rifampin and mupirocin showed good activities, with ranging resistance between 0 and 6.8%. Higher resistance was observed for spiramycin and fosfomycin. Tylosin, vancomycin, metronidazole, dapsone and tiamulin, to which strains showed high rates of resistance, were ineffective. Cephalosporins (especially the third-generation cephalosporins) and quinolones (especially the fluorinated quinolones) were the most effective antimicrobial agents against P. multocida subsp. multocida strains and they might be of value for in vivo use.

Animals↗

Cell surface characteristics and virulence in mice of Pasteurella multocida.

The virulence of three avian strains of Pasteurella multocida was evaluated in mice. Strains P-1059I (serotype A:3) and its uncapsulated variant P-1059B and strain 2723 (serotype A:16) were compared. Capsular material thickness after polycationic ferritin labelling of dextrose starch agar (DSA)-grown P. multocida was shown to vary with the strain and was not always related to virulence. Addition of alpha,alpha' bipyridyl (BIP) (160 microM) to the culture medium did not affect capsule production but increased virulence of strains P-1059B and 2723. None of the strains tested showed dermonecrotic activity. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), outer membrane protein (OMP) profiles indicated for strains P-1059I and P-1059B three proteins of 30, 35, and 38 KDa with the 30 KDa protein being the major one. Strain 2723 showed the same OMP profile but the 38 KDa protein was the major one. DSA + BIP-grown strains showed the same OMP profiles. Whole cell profiles were similar for all strains tested. However, addition of BIP to the culture media increased the virulence of strains P-1059B and 2723 and for all strains a 39 KDa protein was induced by the iron chelator. The results indicate that encapsulation may be important for virulence, but other surface components such as OMPs may be required as well.

Animals↗

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing.

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Animals↗

Pasteurella multocida subdural empyema: a case report.

The first case of Pasteurella Multocida subdural empyema is reported. This was secondary to hematogenous spread from the pharynx to a chronic subdural hematoma. Positive cultures were obtained from the primary source, the blood and the subdural space. The patient was discharged with no neurological deficit following surgical drainage and parenteral antibiotics. The incidence, predisposing factors, clinical features, bacteriology, diagnostic procedures, treatment and mortality of subdural empyema are briefly discussed.

Brain Abscess↗

Repromicin derivatives with potent antibacterial activity against Pasteurella multocida.

Reductive amination of repromicin with polyfunctional amines has led to new macrolide antibacterial agents, some of which are highly potent against the Gram-negative pathogen Pasteurella multocida both in vitro and in a mouse infection model. A key element in this discovery was the recognition that among certain known macrolides increasing lipophilicity results in diminished in vivo activity. One repromicin derivative, 20-[N-[3-(dimethylamino)-propyl]-N-L-alanylamino]-20-deoxorepro micin (35), was selected for advanced evaluation. At 5 mg/kg, a single subcutaneous dose was found to control induced pasteurellosis in swine and induced respiratory disease in cattle.

Animals↗

Quantitative structure-activity relationships among macrolide antibacterial agents: in vitro and in vivo potency against Pasteurella multocida.

Quantitative structure-activity relationships have been found among macrolide antibacterial agents in their potencies against the bacterial pathogen Pasteurella multocida both in vitro and in mouse infections. To obtain these relationships we measured, among other things, the pK(a)'s and log P's of 15 known macrolides of diverse structures. Among these compounds, in vitro potency [log(1/MIC)] is a function of log P, log D, and CMR (R = 0.86). In vivo potency is a function of the higher pK(a), the HPLC chromatographic capacity factor log k', log(1/MIC) and pNF (R = 0.93). pNF is defined as the negative logarithm of the fraction of neutral drug molecules present in aqueous solution at pH 7.4. The same physical properties were determined for 14 macrolides not used in developing the original QSAR models. Using the in vivo model, we calculated the mouse protection potency ranges for these new compounds. Ten estimates agreed with those observed, three were lower by a half-order of magnitude, and one was calculated to be active in the range of 15-50 mg/kg, but in fact was not active at 50 mg/kg, the highest level tested. When these new compounds were combined with the original 15, and the QSAR's updated, the new equations for the in vitro and in vivo potencies were essentially the same as those originally found. Hence, the physical properties indicated above are major determinants of macrolide antibacterial potencies.

Animals↗

Antibiotic sensitivity patterns among Indian strains of avian Pasteurella multocida.

An investigation was carried out to study the antibiotic sensitivity of avian strains of Pasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India. A total of 123 strains of P. multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics. Absolute resistance was observed against sulfadiazine. The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%). The majority of the strains were found to exhibit intermediate sensitivity. Chloramphenicol was selected and suggested for treatment. Antibiogram studies also revealed the emergence of multidrug-resistant strains of P. multocida among Indian poultry.

Animals↗

Cloning and sequencing of a 16 kDa outer membrane protein gene of Pasteurella multocida P52.

The outer membrane proteins (OMPs) of Pasteurella multocida are potential immunogens. The 16 kDa OMP of P. multocida P52, serotype B:2, was identified as one of the major immunodominant antigens. The gene omp16, encoding a 16 kDa outer membrane protein, was amplified, cloned into a pBluescript SK(-) vector and sequenced. Complete sequence homology was observed between the 16 kDa OMP gene of P. multocida P52 (serotypes B:2) and P. multocida T16 (somatic serotype 3,4). This gene was distributed among different serotypes of P. multocida and found to localize in a 6.0 kb HindII fragment of the P. multocida genome.

Bacterial Outer Membrane Proteins↗

Molecular variability among strains of Pasteurella multocida isolated from an outbreak of haemorrhagic septicaemia in India.

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.

Animals↗

Prevalent serotypes of Pasteurella multocida isolated from different animal and avian species in India.

Identification and estimation of the prevalence of Pasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out. Out of 418 samples collected from different outbreaks suspected to be caused by P. multocida, a total of 206 bacterial cultures were identified as P. multocida on the basis of cultural, morphological and biochemical characteristics. All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer. Serotyping of P. multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1 and -:1) among the livestock population. P. multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of approximately 460 bp specific for P. multocida. The results of PCR assay correlated well with conventional methods of identification. The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species.

Animals↗

Lack of effect of aerial ammonia on atrophic rhinitis and pneumonia induced by Mycoplasma hyopneumoniae and toxigenic Pasteurella multocida.

The objective of this experimental study was to determine the effects of aerial ammonia on disease development and bacterial colonization in weaned pigs inoculated with toxigenic Pasteurella multocida and Mycoplasma hyopneumoniae. Two groups of 10 pigs each were continuously exposed to 50 and 100 p.p.m. ammonia, respectively, and compared to a non-exposed control group of 20 pigs. Following aerosol inoculation with M. hyopneumoniae at day 9, all pigs were aerosol-inoculated with toxigenic P. multocida type A at days 28, 42 and 56. At day 63 they were euthanized. Clinical signs including coughing and respiratory distress were present in all groups following inoculation. No significant differences could be established in the extent or frequency of pneumonia between ammonia-exposed pigs and controls, or in the extent of conchal atrophy, the frequency of isolation of toxigenic P. multocida from conchae, tonsils, lungs and kidneys, or the average daily weight gain. The recovery of toxigenic P. multocida from nasal swabs following inoculation was significantly greater in pigs exposed to 50 p.p.m. ammonia or more as compared to the control group. In conclusion, high levels of ammonia combined with inoculations with M. hyopneumoniae and toxigenic P. multocida had no significant effect on disease development, but may have enhanced colonization by toxigenic P. multocida on the nasal turbinates.

Administration, Inhalation↗

Antimicrobial susceptibility of Pasteurella multocida isolated from cattle and pigs.

Minimum inhibitory concentrations (MICs) of 10 antimicrobial agents were determined for Pasteurella multocida from cattle and pigs (72 and 68 isolates, respectively). Higher MICs were observed with oxytetracycline, doxycycline, tilmicosin and thiamphenicol for porcine isolates than for bovine isolates. Enrofloxacin was the most active, with an MIC for 90% of the isolates (MIC90) of 0.05 microg/ml for both bovine and porcine isolates. Aspoxicillin exhibited the same excellent activity against penicillin-susceptible isolates as ceftiofur, with MICs ranging from < or = 0.025 to 0.1 microg/ml. Aminoglycosides were less active, with an MIC90 of > 100 microg/ml for both bovine and porcine isolates.

Amoxicillin↗

Treatment of experimentally induced Pasteurella multocida infections in broilers and turkeys: comparative studies of different oral treatment regimens.

Experimental fowl cholera was induced in 60 healthy 10-week-old broiler chickens and 8-week-old turkeys by intramuscular inoculation with approximately 80 colony-forming units (cfu) of Pasteurella multocida X-73 strain and with approximately 70 cfu of P. multocida P-1059 strain, respectively. This method of infection proved to be useful for evaluating the efficacy of anti-microbial medication, by measuring mortality, weight gain, pathological responses and frequency of re-isolation of P. multocida. The efficacies of two different dosing methods, continuous and pulse dosing, were compared. Using the continuous-dosing method, norfloxacin was administered to drinking water at 100 mg/l for 5 days in chickens. Efficacies were slightly improved compared with pulse dosing at 15 mg/kg bodyweight for the same length of time. The opposite was observed in turkeys, to the degree of control of mortality and maintenance of weight gain.

Administration, Oral↗

Pasteurella gallinarum neonatal meningitis.

A 4-day-old baby weighing 1.7 kg was admitted to the neonatal intensive care unit of Ga-Rankuwa Hospital, Pretoria, with a history of apneic attacks. On examination there was an umbilical sepsis and the neonate was septicemic. The baby had been delivered at home and the umbilical cord had been cut by the grandmother using unclean scissors and chimney soot applied to the umbilical stump. On admission, a septic screen was done and antibiotic treatment was started with penicillin and amikacin. The investigations showed that the baby was slightly anemic, with hemoglobin levels of 10.0 g/dL (14.9-23.7 g/dL), and a pure growth of a Gram-negative bacillus was obtained from the cerebrospinal fluid, blood culture and suprapubic aspirate urine specimens. The Gram-negative bacillus was catalase and oxidase positive and it was identified as Pasteurella gallinarum. Antimicrobial profiling showed the organism to be susceptible to penicillin, cefotaxime, gentamicin and amikacin. Despite having received antimicrobial agents to which the etiological agent was susceptible, the neonate died within 5 days of admission. The cause of death was postulated to be due to overwhelming sepsis which resulted in septic shock.

Bacteremia↗

Development of a 23S rRNA-based PCR assay for the identification of Pasteurella multocida.

AIMS: The aim of this work was to develop a rapid diagnostic test for Pasteurella multocida. METHODS AND RESULTS: A polymerase chain reaction (PCR) assay using primers derived from the 23S rRNA gene sequence of Past. multocida was developed. The PCR assay correctly identified all 144 isolates of Past. multocida tested, including type strains of the three subspecies as well as the reference strains for the Heddleston and Carter typing schemes. Of 20 closely related bacteria from the family Pasteurellaceae tested, only the type strains of Past. canis biovar 2 and Past. avium biovar 2 were positive. These two bacteria, formerly known as Bisgaard Taxon 13, are the closest phylogenetic relatives of Past. multocida based on 16S ribosomal rRNA. All phylogenetically unrelated avian and porcine organisms tested were negative. CONCLUSION: This PCR enables rapid identification of Past. multocida colonies from avian or porcine origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Veterinary diagnostic laboratories can use this PCR to rapidly and accurately diagnose fowl cholera and porcine pasteurellosis.

Animals↗

Pathophysiological changes occurring during Escherichia coli endotoxin and Pasteurella multocida challenge in piglets: relationship with cough and temperature and predicitive value for intensity of lesions.

The aims of this study were (1) to correlate cough and body temperature (BT) with the severity of bronchopneumonia in pigs, (2) to determine whether these clinical signs can be used to early diagnose bronchopneumonia and (3) to assess the predictive values of cough and BT regarding lung lesions. Bronchopneumonia was induced by administering E. coli endotoxin (LPS) combined with Pasteurella multocida type A (PmA) in the trachea of 13 piglets. Saline-instilled negative controls (n = 8), PmA inoculated (n = 6) and LPS instilled (n = 5) groups were also constituted. Cough and BT were recorded daily while the bronchopneumonia severity was assessed using bronchoalveolar lavage fluid (BALF) cytology, cytokines and measurement of lung lesion volume. Changes in expiratory breathing pattern were also measured (Penh). The combination of LPS and PmA induced a subacute bronchopneumonia characterised by macrophage, neutrophil, and lymphocyte infiltration, changes in Penh and an increase in the mRNA level of IFN-gamma while IL8, IL-18 and TNF-alpha mRNA levels remained unchanged. The daily body weight gain of infected animals was significantly reduced. Cough and BT changes were proportional to the intensity of the lung inflammatory process, functional respiratory changes and to the extent of macroscopic lesions. When comparing the individual values of cough and BT to thresholds defined for both parameters, an early diagnosis of pneumonia was possible. Considering the pooled data of each group, it was possible to define thresholds allowing an early segregation between the groups of diseased and healthy piglets. The daily values of cough and BT were predictive for the volume of lung lesions recorded at the end of the trial. In conclusion, cough and BT appear as potential indicators for the intensity and the evolution of the respiratory disease. They also seem to be good predictors for the magnitude of lung lesions and weight gain recorded at the study endpoint.

Animals↗