The Role of Necroptosis, Apoptosis, and Inflammation in Fowl Cholera-Associated Liver Injury in a Chicken Model.

Fowl cholera resulting from infection with Pasteurella multocida causes huge economic losses in the poultry industry. Necrotic hepatitis is reported to be a significant lesion associated with fowl cholera in chickens. Clarifying the underlying molecular mechanism of hepatic injury caused by P. multocida infection is needed to develop new strategies to control fowl cholera. Pasteurella multocida Q (the standard reference strain) and P. multocida 1G1 (a clinical strain) were used to infect healthy laying hens. Clinical signs were observed and gross lesions in livers were observed postmortem. Histologic lesions and the localization and expression of protein molecules associated with necroptosis, apoptosis, and inflammation in hepatic tissues were examined by hematoxylin and eosin staining and immunohistochemistry. Western blot analysis was used to determine the expression of liver injury-related genes. Necroptotic molecules such as RIPK1 (receptor interaction protein kinases 1), RIPK3 (receptor interaction protein kinases 3), and MLKL (mixed lineage kinase domain-like protein) were observed by immunostaining primarily in the cytoplasm of hepatocytes within or around necrotic foci, and inflammatory mediators HMGB1 (high-mobility group box 1) and IL-6 (interleukin-6) were found in the cytoplasm of heterophils, monocytes/macrophages, and hepatic sinusoids. In addition, MMP9 (matrix metalloproteinase 9) and TIMP1 (tissue inhibitor of metalloproteinase 1) were observed in hepatic parenchymal cells, inflammatory cells, and interstitial spaces, whereas the apoptotic effector molecule caspase-3 (cysteine-containing aspartic proteolytic enzymes 3) was mainly found in hepatocytes. The expression of RIPK1, RIPK3, and MLKL was significantly higher in the infected chickens than in the controls. HMGB1 and IL-6 protein levels were also increased in infected chickens relative to those in controls. Both MMP9 and TIMP1 were highly expressed in infected chickens. In addition, caspase-3 protein levels were significantly elevated in infected chickens. Necroptosis, apoptosis, and inflammation played a significant role in hepatic injury caused by P. multocida.

Clinical Features and Outcomes of Pasteurella multocida Infection.

Pasteurella multocida, a zoonotic infectious organism, has most often been described in patients after an animal bite. Here, we characterize the clinical features and outcomes of P multocida infection in a large cohort of patients according to the presence or absence of an animal bite.We retrospectively searched MUSC's laboratory information system for all patients with positive P multocida cultures from 2000 to 2014. Extensive data were abstracted, including clinical and outcome data. The Charlson comorbidity index (CCI) was used to assess comorbidities among patients.We identified 44 patients with P multocida infections, including 25 with an animal bite. The average age was 64 years and the majority of patients were women (N = 30). There was no difference in age and sex distribution among those with and without a bite (P = 0.38 and 0.75, respectively). A CCI ≥1 was significantly associated with the absence of a bite (P = 0.006). Patients presenting without a bite were more frequently bacteremic (37% vs 4%, respectively, P = 0.001), and were hospitalized more often (84% vs 44%, respectively, P = 0.012). Of the 8 patients who required intensive care unit (ICU)-based care, 7 were non-bite-related. There were 4 deaths, all occurring in patients not bitten.P multocida infections not associated with an animal bite were often associated with bacteremia, severe comorbidity(ies), immune-incompetent states, the need for ICU management, and were associated with substantial mortality.

Feather corticosterone reveals effect of moulting conditions in the autumn on subsequent reproductive output and survival in an Arctic migratory bird.

For birds, unpredictable environments during the energetically stressful times of moulting and breeding are expected to have negative fitness effects. Detecting those effects however, might be difficult if individuals modulate their physiology and/or behaviours in ways to minimize short-term fitness costs. Corticosterone in feathers (CORTf) is thought to provide information on total baseline and stress-induced CORT levels at moulting and is an integrated measure of hypothalamic-pituitary-adrenal activity during the time feathers are grown. We predicted that CORTf levels in northern common eider females would relate to subsequent body condition, reproductive success and survival, in a population of eiders nesting in the eastern Canadian Arctic during a capricious period marked by annual avian cholera outbreaks. We collected CORTf data from feathers grown during previous moult in autumn and data on phenology of subsequent reproduction and survival for 242 eider females over 5 years. Using path analyses, we detected a direct relationship between CORTf and arrival date and body condition the following year. CORTf also had negative indirect relationships with both eider reproductive success and survival of eiders during an avian cholera outbreak. This indirect effect was dramatic with a reduction of approximately 30% in subsequent survival of eiders during an avian cholera outbreak when mean CORTf increased by 1 standard deviation. This study highlights the importance of events or processes occurring during moult on subsequent expression of life-history traits and relation to individual fitness, and shows that information from non-destructive sampling of individuals can track carry-over effects across seasons.

Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice.

Pasteurella pneumotropica is an opportunistic pathogen in rodents. Natural infection in immunodeficient animals suggests that immunodeficiency is a major factor in P. pneumotropica pathogenesis. To understand this process, we performed clinical, pathological and bacteriological studies of immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice experimentally infected with P. pneumotropica ATCC 35149. From 14 days postinoculation, some of P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of weight loss. Three of 10 P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of depression, ruffled coat, and weight loss and died at 27, 34, and 59 days postinoculation. At 35 days postinoculation, almost all P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice had lung abscesses. The bacteria were isolated from the upper and lower respiratory tracts, including the lungs, and blood. In contrast, P. pneumotropica-infected ICR mice exhibited no clinical signs or lesions. The bacteria were isolated from the upper, but not the lower respiratory tracts. We developed an animal model for understanding host interactions with P. pneumotropica.

Identification of novel glycosyltransferases required for assembly of the Pasteurella multocida A:1 lipopolysaccharide and their involvement in virulence.

We previously determined the structure of the Pasteurella multocida Heddleston type 1 lipopolysaccharide (LPS) molecule and characterized some of the transferases essential for LPS biosynthesis. We also showed that P. multocida strains expressing truncated LPS display reduced virulence. Here, we have identified all of the remaining glycosyltransferases required for synthesis of the oligosaccharide extension of the P. multocida Heddleston type 1 LPS, including a novel alpha-1,6 glucosyltransferase, a beta-1,4 glucosyltransferase, a putative bifunctional galactosyltransferase, and two heptosyltransferases. In addition, we identified a novel oligosaccharide extension expressed only in a heptosyltransferase (hptE) mutant background. All of the analyzed mutants expressing LPS with a truncated main oligosaccharide extension displayed reduced virulence, but those expressing LPS with an intact heptose side chain were able to persist for long periods in muscle tissue. The hptC mutant, which expressed LPS with the shortest oligosaccharide extension and no heptose side chain, was unable to persist on the muscle or cause any disease. Furthermore, all of the mutants displayed increased sensitivity to the chicken antimicrobial peptide fowlicidin 1, with mutants expressing highly truncated LPS being the most sensitive.

Decoration of Pasteurella multocida lipopolysaccharide with phosphocholine is important for virulence.

Phosphocholine (PCho) is an important substituent of surface structures expressed by a number of bacterial pathogens. Its role in virulence has been investigated in several species, in which it has been shown to play a role in bacterial adhesion to mucosal surfaces, in resistance to antimicrobial peptides, or in sensitivity to complement-mediated killing. The lipopolysaccharide (LPS) structure of Pasteurella multocida strain Pm70, whose genome sequence is known, has recently been determined and does not contain PCho. However, LPS structures from the closely related, virulent P. multocida strains VP161 and X-73 were shown to contain PCho on their terminal galactose sugar residues. To determine if PCho was involved in the virulence of P. multocida, we used subtractive hybridization of the VP161 genome against the Pm70 genome to identify a four-gene locus (designated pcgDABC) which we show is required for the addition of the PCho residues to LPS. The proteins predicted to be encoded by pcgABC showed identity to proteins involved in choline uptake, phosphorylation, and nucleotide sugar activation of PCho. We constructed a P. multocida VP161 pcgC mutant and demonstrated that this strain produces LPS that lacks PCho on the terminal galactose residues. This pcgC mutant displayed reduced in vivo growth in a chicken infection model and was more sensitive to the chicken antimicrobial peptide fowlicidin-1 than the wild-type P. multocida strain.

Diabetes in patients with pasteurellosis.

From 1991 to 2003, 20 patients with pasteurellosis were admitted to our unit, of whom 2 died. They presented with cellulitis (n = 14), arthritis (n = 6), pneumonia (n = 3), subcutaneous abscess (n = 3), bursitis (n = 2), meningitis, otitis, sinusitis and uveitis. Underlying diseases included diabetes (n = 6) and malignancy (n = 5). Diabetes could be a predisposing condition for pasteurellosis.

Construction and virulence of a Pasteurella multocida fhaB2 mutant in turkeys.

Pasteurella multocida is the causative agent of fowl cholera. The organism can occur as a commensally in the naso-pharyngeal region of apparently healthy animals and it can be a primary or secondary pathogen in the disease process of birds. The complete genome of an avian strain of P. multocida has been sequenced and was shown to possess two filamentous hemagglutinin genes designated fhaB1 and fhaB2. Filamentous hemagglutinin transposon mutants of a bovine strain of P. multocida are attenuated in mice. Here, we report the construction of an fhaB2 P. multocida mutant in an avian strain P-1059 (A:3). The fhaB2 mutant and the parent were assessed for virulence in turkeys by intranasal and intravenous challenge. Inactivation of fhaB2 resulted in a high degree of attenuation when turkeys were challenged intranasally and to a lesser degree when intravenously administered. Resistance of the fhaB2 mutant and parent strain to killing by serum complement was similar.

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.

Septicaemia and arthritis in pigs experimentally infected with Pasteurella multocida capsular serotype A.

Twenty-five caesarean-derived, colostrum-deprived (CDCD) pigs and 18 specific pathogen-free pigs, aged 8 to 14 weeks, were inoculated intranasally or intratracheally with Pasteurella multocida capsular serotype A, isolated from a severe pneumonic lesion in a growing pig. The pigs were killed for necropsy on day 6 or 14 post-inoculation (PI) or, in the case of the only fatally infected animal, examined at the time of death. One CDCD pig, inoculated intratracheally with 5 ml of a bacterial suspension containing 1.7x10(9) colony-forming-units/ml, died of septicaemia on day 1 PI. Histological lesions such as severe pleuropneumonia, thrombi in glomerular capillaries, haemorrhage of the spleen, and abscesses in the tonsillar crypts were observed. The organism was recovered from a number of sites and its antigens were detected immunohistochemically in the pneumonic lesions, blood vessels of the tissues, and tonsillar crypts in the dead pig. Pneumonia, pleural adhesions and suppurative arthritis in the extremital joints were observed grossly in 3/29, 8/29 and 7/29 intratracheally inoculated pigs, respectively. In intranasally inoculated pigs, no macroscopical abnormalities were seen; histologically, however, exudative bronchopneumonia and fibrinous pleurisy were observed in 9/14 and 4/14 pigs, respectively. No significant changes were seen in the tissues of uninfected control pigs. The organism was recovered from the lesions and P. multocida type A antigen was demonstrated immunohistochemically. The organism was rarely recovered from the liver, spleen or lymph nodes (bronchopulmonary or mesenteric). The results suggest that P. multocida capsular serotype A alone can cause not only pneumonia in pigs but also septicaemia or arthritis.

Acute disseminated encephalomyelitis associated with Pasteurella multocida meningitis.

OBJECTIVE: To describe a case of Pasteurella multocida meningitis associated with acute disseminated encephalomyelitis (ADEM). CASE REPORT: A 33-year-old woman employed in a dog pound presented herself to hospital with fever and meningismus and was found to have culture positive Pasteurella multocida meningitis. Despite appropriate antibiotic treatment her clinical course was characterized by a persistent fever and worsening encephalopathy, which prompted further neurological investigation. Spinal fluid exam and serial MRI scans as well as her one-year clinical course were found to be compatible with ADEM. CONCLUSION: Persistent fever and worsening encephalopathy in meningitis may indicate a para-infectious immune process such as ADEM, and may serve as indications for further neurological investigation.

Pasteurella multocida urinary tract infection with molecular evidence of zoonotic transmission.

We describe a patient with a urinary tract infection (UTI) caused by Pasteurella multocida. Pulsed-field gel electrophoresis revealed that the clinical isolate recovered from the patient was identical (100% band match) to P. multocida isolates recovered from the patient's cat, but the isolate differed from an isolate recovered from a visiting cat and from a laboratory control strain. The patient also had abnormal urologic anatomy secondary to surgery; this has also been associated with P. multocida UTI.

Serum resistance of Pasteurella multocida in avian and porcine sera, and comparative virulence investigations of selected serum-sensitive and resistant strains in chickens.

Growth in serum of Pasteurella multocida and related species in chicken, turkey, duck and pig sera were compared, and selected serum-resistant and serum-sensitive strains were inoculated into 18-week-old layers. Eighty-seven field strains of Pasteurella spp. and nine reference strains representing different clones defined by restriction endonuclease analysis (REA) profiles were used in the study. Serum activity was measured by changes in the optical density (OD) of the serum after inoculation and incubation at 41 degrees C for chicken, turkey and duck serum and 39 degrees C for pig serum. Serum activity was measured by comparison with previously determined serum-resistant (P-1059) and serum-sensitive (CU vaccine) strains, and classified into highly serum-resistant, moderately serum-resistant and serum-sensitive. Strains of the same REA type were found to have identical growth curves and the same maximum OD values when tested in serum from the same host species. Turkey serum was shown to be less inhibitory to a wide range of P. multocida strains than chicken, duck and pig sera. Serum-resistant strains were demonstrated among avian as well as mammalian strains. Among the avian strains, the proportion of serum-resistant strains was higher in outbreak strains than in strains from apparently healthy carriers. Removal of the capsule from selected strains by hyaluronidase treatment failed to change the serum activity. The most severe lesions in experimentally infected chickens were produced by a serum-resistant strain; however, lesions were also found in chickens infected by serum-sensitive strains, indicating the involvement of multiple factors in the virulence of P. multocida. Further investigations on serum resistance are indicated in order to relate other host and bacterial factors responsible for the development of fowl cholera.

Taxonomic subgroups of Pasteurella multocida correlate with clinical presentation.

Pasteurella multocida is a small nonmotile gram-negative coccobacillus that is found in the nasopharynx and gastrointestinal tract of many wild and domesticated animals. In humans it most commonly causes cellulitis and localized superficial skin abscesses following an animal bite or scratch. The respiratory tract is the second most common site of infection for PASTEURELLA: Of the more than 17 species of Pasteurella known, Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica are among the most common pathogens in humans. With the use of molecular techniques, distinction between different subspecies of P. multocida can be made more easily and accurately. We used the sequence of the 16S ribosomal DNA (rDNA) and repetitive extragenic palindromic sequence-PCR (REP-PCR) to characterize 20 strains (14 of P. multocida subsp. multocida and 6 of P. multocida subsp. septica; the 16S rDNA is identical for P. multocida subsp. multocida and Pasteurella multocida subsp. gallicida but differs from that of P. multocida subsp. septica) isolated from various anatomic sites. We found excellent correlation between the 16S rDNA sequence (a marker for a small conserved region of the genome), REP-PCR (a marker for a large portion of the genome), and biochemical tests (trehalose and sorbitol). We also found a correlation between the clinical presentation and the taxonomic group, with P. multocida subsp. septica more often associated with wounds than with respiratory infections (67 versus 17%, respectively) (P < 0.05, Z test) and P. multocida subsp. multocida more often associated with respiratory infections than with wounds (71 versus 14%, respectively) (P < 0.05, Z test).

Pharmacokinetic and pharmacodynamic profiles of danofloxacin administered by two dosing regimens in calves infected with Mannheimia (Pasteurella) haemolytica.

The pharmacokinetics and pharmacodynamics of danofloxacin in calves with induced Mannheimia (Pasteurella) haemolytica pneumonia were evaluated. Calves received either saline as an intravenous (IV) bolus or danofloxacin (0.738 mg/kg of body weight) administered as either a single IV bolus or a 36-h continuous IV infusion. Blood samples and bronchial secretions were collected before and at predetermined times over 48 h following the start of treatment. Calves were assessed clinically throughout, and lung consolidation was assessed at necropsy. Bronchial secretions and lung tissue were cultured for M. haemolytica. Bolus administration of danofloxacin produced a high maximum drug concentration-to-MIC ratio (C(max):MIC) of 14.5 and a time period of 9.1 h when plasma danofloxacin concentrations exceeded the MIC (T>MIC). Following danofloxacin infusion, the C(max):MIC was low (2.3), with a long T>MIC (33.3 h). The area under the curve-to-MIC ratios were 43.3 and 49.1 for the bolus and infusion administrations, respectively. The single bolus of danofloxacin was more effective than the same dose administered by continuous infusion, as indicated by a significantly lower (P < 0.05) number of animals with M. haemolytica in bronchial secretions after treatment and lower rectal temperatures in the 24 h after the start of treatment. Thus, danofloxacin exhibited concentration-dependent antimicrobial activity in cattle with respiratory disease caused by M. haemolytica.

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.