Investigations into an outbreak of corvid respiratory disease associated with Pasteurella multocida.

The possible cause of disease and mortality in corvids on an outdoor pig unit in the north of England between August 2007 and March 2008 was investigated. Nine carrion crows (Corvus corone corone) and nine rooks (Corvus frugilegus), comprising five live-caught birds with clinical signs of respiratory disease, one live-caught bird without respiratory disease, and 12 birds submitted dead were examined. Clinical signs, gross and histopathological examination, microbiology and toxicology indicated that Pasteurella multocida infection was the cause of disease. Molecular and serotyping analyses showed that P. multocida isolates (obtained from live-caught birds with clinical respiratory disease) were all capsular type F with a mix of somatic serotypes 3, 4 and 7. Immunohistochemistry increased the diagnostic sensitivity of the analysis and detected P. multocida within the pulmonary lesions of all affected live-caught birds and 10 of 12 birds found dead. These findings suggest that wild corvids in the UK can suffer from lung pathology associated with P. multocida and, as potential vectors of P. multocida, may pose a risk to domestic poultry.

Contribution of respiratory burst activity to innate immune function and the effects of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro.

The mechanisms of interaction between phagocytes and different bacteria that help resolve lung infections or contribute to lung pathology are poorly defined. Alveolar phagocytes (resident macrophages and recruited neutrophils) make a major contribution to innate immunity by mounting a respiratory burst that helps kill internalised bacteria. However, this ability may be altered during or after exposure to infection. This review considers the application and limitations of a variety of analytical methods for oxygen-dependent mechanisms of respiratory burst in phagocytes initiated by soluble and particulate activators. Particular reference is given to the study in vitro of phagocytes from healthy and diseased ruminants during either natural infection with Mycobacterium avium paratuberculosis or experimental infection with Pasteurella multocida or Mannheimia haemolytica.

Endotoxin and mammalian host responses during experimental disease.

Endotoxin is an integral component of the outer membrane of Gram-negative bacteria and a prime example of unique and highly conserved bacterial surface molecules that engage with the innate immune system of the mammalian host via pattern recognition receptors on a range of host cells. The results of this interaction, which may be beneficial or detrimental to the development and welfare of the host, are reviewed, focusing on the different sensitivities and consequences in a range of hosts of experimental exposure to endotoxin, the disease outcomes and recent developments in our understanding of the mechanisms involved.

Differences in Virulence Between Bovine-Derived Clinical Isolates of Pasteurella multocida Serotype A from the UK and the USA in a Model of Bovine Pneumonic Pasteurellosis.

The time of onset and subsequent degree and progression of clinical signs, bacterial colonization and tissue pathology during experimental disease induced by intratracheal inoculation of either a UK or USA isolate of Pasteurella multocida serotype A recovered from clinical cases of bovine pneumonia were determined. Calves aged 8 weeks were challenged with 300 ml phosphate buffered saline (PBS) alone (group 1, n = 3, negative control) or containing 7.1 × 10(8) colony forming units (cfu) of UK isolate (group 2, n = 8) or 5.8 × 10(8) cfu of USA isolate (group 3, n = 8). Bronchoalveolar lavage (BAL) at 0, 1 and 4 days post challenge (dpc) and at the time of necropsy examination (7-8 dpc) showed no significant differences between groups 2 and 3 in bacterial numbers recovered. No P. multocida were recovered from group 1 animals. No clinical disease was present in group 1 calves and in group 3 was limited to scour in 1 calf at 1 dpc. All calves in group 2 had reduced food intake at 4-5 dpc, five had periods of dullness, three a mild nasal discharge at 1 dpc, four had mild to substantial respiratory stridor and one was killed at 6 dpc for humane reasons. Rectal temperatures remained about 39°C in group 1 calves, but increased in P. multocida-challenged calves to 40-41°C within 8-12 h of challenge. Significantly (P = 0.01) greater percentages of lung surface area were consolidated in group 2 (mean ± SD, 21 ± 10.1) compared with group 3 (7 ± 8.6) calves. Significantly more extensive and severe histological lesions were present in the lung lobes (P = 0.006) and lymph nodes (P = 0.02) of group 2 compared with group 3 calves. Pleurisy was present in group 2 calves only and no pathology was present in group 1. Pulsed-field gel electrophoresis (PFGE) produced 11 (group 2, UK isolate) or 10 (group 3, USA isolate) bands with differences in banding patterns. Results overall showed that two isolates, distinct geographically and genetically (by PFGE), caused pneumonic pasteurellosis in a single host with significantly different severity of pathology. This information is relevant to the development of novel vaccine control and interpretation of diagnostic results.

Vector densities that potentiate dengue outbreaks in a Brazilian city.

To identify the critical vector density that potentiates dengue outbreaks in an endemic site and to identify obstacles to anti-dengue activities, we correlated a series of dengue outbreaks in a Brazilian city with the intensity of its anti-vector source-reduction activities. The proportion of houses infested by vector mosquitoes correlated inversely with intensity of anti-mosquito interventions, and the vector population developed independently of rainfall. Local periods of drought promoted vector abundance in two ways: residents stored water in which vector mosquitoes could breed, and cholera outbreaks due to contaminated water diverted local health workers from routine anti-vector activities. One dengue outbreak became apparent to authorities more than two months after it commenced but would have been identified almost immediately had dengue-like disease in indicator hospitals been monitored. Active surveillance, therefore, offers a window of opportunity for promptly executed anti-dengue interventions. Source-reduction measures that suppress vector infestations to less than 1% of houses effectively avert outbreaks of dengue.

Prophylactic use of human endotoxin-core hyperimmune gammaglobulin to prevent endotoxaemia in colostrum-deprived, gnotobiotic lambs challenged orally with Escherichia coli.

The efficacy of human IgG polyclonal antibody to endotoxin-core in preventing endotoxaemia and subsequent disease was studied in colostrum-deprived gnotobiotic lambs challenged orally at about 5 h old with 10(9) cfu Escherichia coli. Human endotoxin-core hyperimmune gammaglobulin was given intravenously to 5 lambs at 1.9 g IgG/kg bodyweight prior to challenge. Human albumin was given intravenously to 3 control lambs. Bacteraemia was observed in all lambs, but the incidence was lower (P < 0.01) and the onset later (P < 0.05) in gammaglobulin pre-treated lambs. These lambs showed no signs of disease, whereas clinical endotoxaemia, manifesting as watery mouth disease, was diagnosed in 2 of the 3 control lambs which were killed between 18 and 22 h after challenge. Thus, prophylactic treatment of colostrum-deprived lambs with human IgG enriched in endotoxin-core antibodies was effective in reducing the degree of bacteraemia and preventing endotoxaemia, leukopenia and clinical disease following oral challenge with E. coli.

Interrupted blood-feeding by Culiseta melanura (Diptera: Culicidae) on European starlings.

To determine whether Culiseta melanura (Coquillett) mosquitoes tend to take multiple blood meals when birds of certain species serve as hosts, we compared the frequencies with which such mosquitoes fed upon caged starlings and robins and determined whether similar volumes of blood were imbibed from each. The blood of robins (Turdus migratorius) and European starlings (Sturnus vulgaris) was marked contrastingly by injecting birds with rubidium or cesium salts. Caged birds were placed together in a natural wetland setting overnight. Mosquitoes captured nearby on the following morning were analyzed for each of the elemental markers. Where marked robins and starlings were equally abundant, 43% of freshly engorged Cs. melanura fed on more than or equal to two hosts. More Cs. melanura fed on robins than on starlings. Individual mosquitoes tended to contain far more robin- than starling-associated marker, indicating that mosquitoes "feasted" on robins but only "nibbled" on starlings. Mosquitoes marked with both elements apparently fed meagerly on the starlings then abundantly on the robins. Our estimates of bloodmeal volume indicate that 85% of mosquitoes that fed on marked starlings obtained < 0.5 microliter of blood from them. We suggest that defensive behavior by starlings interrupts mosquito blood-feeding and that, in a communal roost of starlings, each mosquito will tend to feed on more than one bird, thereby promoting rapid transmission of such ornithonotic arboviruses as eastern equine encephalomyelitis virus and West Nile virus.

Pathophysiological and immune cell responses in calves prior to and following lung challenge with formalin-killed Pasteurella multocida biotype A:3 and protection studies involving subsequent homologous live challenge.

Pneumonic pasteurellosis is a common respiratory infection in cattle that has major economic and welfare implications world-wide and the incidence in the UK due to Pasteurella multocida, currently the same as that associated with Mannheimia haemolytica, is increasing. Whereas much is known regarding the pathogenesis of M. haemolytica infections little information is available on the pathogenic process of pasteurellosis initiated by P. multocida. In the present work calf systemic and innate immune responses to intratracheal challenge with formalin-killed P. multocida biotype A:3 and to subsequent experimental lung infection with live P. multocida were investigated. Eight-week-old calves were challenged intratracheally on day 0 with either 10(9) colony forming units (cfu) of formalin-killed P. multocida biotype A:3 in 300 ml saline (n = 10) or 300 ml saline alone (n = 10), followed, at day 21, by challenge with 10(9) cfu live P. multocida. Pathophysiological and lung phagocyte responses were assessed by clinical monitoring, sequential lung lavage and blood sampling. Results for samples obtained before, during and after challenge showed clinical and acute phase protein responses to both bacterial culture and saline control treatments, although higher responses were associated with bacterial challenge. Phagocytosis of P. multocida during 1h incubation periods with lavaged cells in vitro was unaffected by exposure in vivo to killed P. multocida and there was evidence that P. multocida was able to survive intracellularly during this assay. There was no indication that lung exposure to formalin-killed P. multocida conferred protection against subsequent homologous live challenge.

Renal ultrastructure in lamb nephrosis.

Ultrastructural investigation of the kidneys of 18 lambs with clinical nephrosis revealed that groups of tubules showing various degrees of degeneration up to necrosis could exist alongside tubules showing proliferative or regenerative changes. Lesions were most common in proximal convoluted tubules, less common in distal tubules and absent from collecting ducts or straight tubules, though the latter were often distended with hyaline or flocculent casts. The basement membranes of necrotic tubules were intact, suggesting that the potential for regeneration was unimpaired. Nine lambs had lesions in the glomeruli, though the distribution was focal. Glomerular lesions occasionally were acute and degenerative but more usually were proliferative, with occlusion of capillaries due to endothelial swelling and mesangial encroachment. Basement membrane splitting or reduplication was a feature of some glomeruli, but neither subepithelial nor subendothelial dense deposits were seen. The changes were consistent with damage by an unknown nephrotoxic factor or factors.

Predisposition of specific pathogen-free lambs to Pasteurella haemolytica pneumonia by Bordetella parapertussis infection.

Three groups of specific pathogen-free (SPF) lambs were inoculated intratracheally with an ovine isolate of Bordetella parapertussis (5.5 x 10(9) colony-forming units) or with B. parapertussis followed 2 or 5 days later with Pasteurella haemolytica serotype A2 (120-180 million colony-forming units). When P. haemolytica A2 was administered 2 days after infection with B. parapertussis all lambs became febrile for at least 72 h. At necropsy their lungs were discoloured, congested and showed large areas of collapse and consolidation which, in one case, covered the entire lung. Histopathological examination confirmed that the combined infection produced a severe acute bronchopneumonia in four of seven lambs. B. parapertussis and P. haemolytica were recovered from all of the lambs in this group. Seven lambs challenged with P. haemolytica 5 days after B. parapertussis and six lambs infected with B. parapertussis alone showed no clinical signs of disease other than mild pyrexia and only mild histopathological changes. B. parapertussis, but not P. haemolytica, was recovered from these lambs. The findings indicated that B. parapertussis predisposed the SPF lambs to P. haemolytica pneumonia. This effect appeared to be dependent upon the time interval between the administration of the two agents.

Effects of local anesthesia and intravenous naloxone on the changes in behaviour and plasma concentrations of cortisol produced by castration and tail docking with tight rubber rings in young lambs.

The reliability of some behavioral and physiological indices used for the recognition and assessment of acute pain in lambs after castration and tail docking has been examined. Changes in the indices were measured after blocking neural activity with local anaesthetic (lignocaine) and after an opioid antagonist (naloxone) was administered. Six lambs, aged less than one week, were allocated randomly to each of six treatments. (i) control handling and blood sampling; (ii) castration plus tail docking with tight rubber rings; (iii) local anaesthesia; (iv) local anaesthesia followed by castration and tail docking; (v) intravenous naloxone only (0.2 mg kg-1); and (vi) intravenous naloxone followed by castration and tail docking. Local anaesthesia eliminated the behavioural and plasma cortisol changes which usually follow castration and tail docking. Naloxone had a limited effect on the increase in cortisol but altered the behaviour. The results support the view that such indices are useful for assessment of the response to acute pain and that, although endogenous opioids do reduce pain in young lambs after castration and tail docking, the effect is small.

Characterisation of compounds isolated from the sera of horses with acute grass sickness.

Isolates were prepared from the sera of 12 horses with acute grass sickness, using methods reported to yield serum fractions associated with neurotoxicity, and their components identified by liquid chromatography and spectroscopy. All isolates were found to contain cortisol and six isolates also contained a degradation product of an analgesic drug, dipyrone. However, no recognised neurotoxin was detected.

Immediate responses in serum TNF alpha and acute phase protein concentrations to infection with Pasteurella haemolytica A1 in calves.

The concentrations of tumour necrosis factor-alpha (TNF alpha), serum amyloid A (SAA) and haptoglobin were determined in serum samples taken from four calves in the 10 hours after their intra-tracheal inoculation with Pasteurella haemolytica serotype A1. The concentration of haptoglobin did not increase but the concentration of SAA rose progressively from within two hours of inoculation. The concentration of TNF alpha reached a peak in all the animals two hours after inoculation but had returned to undetectable levels after a further four hours. TNF alpha is likely to be an important mediator of the acute phase response in cattle and SAA is a more rapid bovine acute phase protein than haptoglobin in its response to infection with P haemolytica.

Development of a clinical syndrome resembling haemorrhagic septicaemia in the buffalo following intravenous inoculation of Pasteurella multocida serotype B:2 endotoxin and the role of tumour necrosis factor-alpha.

Clinical changes and acute phase responses, including tumour necrosis factor-alpha (tnfalpha), in six buffalo calves were examined following intravenous inoculation of a bolus of endotoxin (1 microg kg(-1) bodyweight in 10 ml of phosphate-buffered saline [ pbs ]) extracted from Pasteurella multocida serotype B:2, the bacterium responsible for haemorrhagic septicaemia (hs) in Asia. Endotoxin injection caused a rapid onset of clinical signs characterised by dullness, sternal recumbency, elevated rectal temperatures, excessive salivation and dyspnoea that lasted for up to 12 hours post-inoculation (p.i.). Serum concentrations of tnfalpha rose within 1 hour p.i. to reach peak values ranging between 8 and 140 ng ml(-1) at 1-2 hours p.i. and then declined rapidly to baseline levels 3-5 hours p.i. Endotoxin injection induced other acute phase changes, including a rapid leucopenia and reductions in the serum concentrations of iron and zinc and a delayed but prolonged increase in haptoglobin from 12 hours p.i. that reached a plateau from about 60 hours p.i. Three control calves injected with 10 ml pbs showed no clinical or blood compositional changes. By reproducing key signs of hs the work confirms a pivotal role of endotoxin in the pathogenesis of hs and emphasises the exquisite sensitivity of the buffalo to P multocida endotoxin.

Biochemical and haematological evidence of endotoxic shock in gnotobiotic lambs with watery mouth disease.

Eight gnotobiotic lambs deprived of colostrum were infected by mouth when two hours old with nonenterotoxigenic strains of Escherichia coli. All developed clinical signs of watery mouth disease and seven died within 24 hours. The mean concentrations of several blood constituents were determined in samples taken at intervals until 24 hours after infection in infected lambs and in four control lambs. The biochemical and haematological changes observed in the lambs developing watery mouth disease were those characteristic of endotoxic shock.

Experimental induction of pneumonic pasteurellosis in calves by intratracheal infection with Pasteurella multocida biotype A:3.

The study aimed to establish an experimental model to investigate the pathogenesis of lung infection by Pasteurella multocida, an important cause of bovine respiratory disease. An experimental model is required to assist the development of an effective vaccine. Sixteen 8-week-old calves were challenged intratracheally with 10(9) or 10(10) colony forming units of P. multocida in either 60 or 300 ml saline in a 2 x 2 factorial experiment. All animals became dull within 2-6h post-infection (p.i.) and two calves were killed humanely because of suspected endotoxic shock. Remaining animals showed increased respiratory rates by 15-20 h p.i. and, at 23 h p.i., calves given the high dose, high volume challenge showed higher (P < 0.05) rectal temperatures. From 24 to 36 h p.i., clinical signs decreased in a majority of animals. Plasma haptoglobin concentrations increased (P < 0.05) in calves given the high volume challenge irrespective of the number of bacteria. At post-mortem examination (4d p.i.), lung lesions, mainly in the apical lobes, were found in all calves. Histopathological examination showed areas of purulent pneumonia with a tendency to abscessation and inflamed interlobular septa characterised by accumulation of neutrophils and oedema. The clinical and pathological responses described were typical of bovine pneumonic pasteurellosis.

Efficacy of a single dose of oral antibiotic given within two hours of birth in preventing watery mouth disease and illthrift in colostrum-deficient lambs.

An antibiotic with a product licence limited to the treatment and control of infectious bacterial enteritis associated with Escherichia coli in piglets was tested for its ability to control watery mouth disease in neonatal lambs. Three groups of lambs were kept in conditions commonly encountered in intensive lambing systems, where high levels of environmental bacterial contamination may be expected. They were allocated at birth to: a control group (group 1) consisting of 18 colostrum-deprived lambs; group 2, consisting of 17 lambs given one feed of colostrum when they were two hours old; and group 3, consisting of 18 colostrum-deprived lambs given spectinomycin orally when they were two hours old. Nine group 1 lambs became diseased and were killed for humane reasons. Blood biochemical changes included hyperglycaemia followed by hypoglycaemia, lactacidaemia, hypoproteinaemia and metabolic acidosis, and postmortem examination of the diseased lambs showed signs consistent with endotoxaemia and a clinical diagnosis of watery mouth disease. Coliforms were isolated from the blood of all group 1 lambs and from half the lambs in groups 2 and 3, but endotoxaemia and watery mouth disease occurred only in group 1 lambs. The results for groups 2 and 3 showed that neither colostrum nor antibiotic at the rates and frequency used prevented bacteraemia, although consecutive samples were positive only on three occasions. Group 3 lambs consistently grew more rapidly than the surviving group 1 lambs and as rapidly as group 2 lambs. There was no evidence that male lambs were more prone to watery mouth disease than female lambs. The results indicated that the antibiotic spectinomycin did not induce endotoxaemia during low-grade bacteraemia and that a single oral dose given within two hours of birth protected colostrum-deprived lambs delivered into a contaminated indoor environment against watery mouth disease.

Acute nephropathy in young lambs.

Acute renal failure was diagnosed by clinical, necropsy and histological criteria in 39 flocks (20 low ground, 13 hill and six marginal upland) in areas served by six veterinary investigation centres. Forty-eight lambs of 12 different breeds or crosses were investigated. The mean age of affected lambs was 38 days (range seven to 84 days); 21 lambs (44 per cent) were aged seven to 28 days, while only eight (17 per cent) were older than two months. Mortality in clinically affected lambs was almost 100 per cent, with no response to various treatments. Histological examination showed that 40 lambs (83 per cent) had nephrosis, while the rest had toxic tubular necrosis, interstitial nephritis or tubular damage associated with oxalate crystal deposits. Only about half of the lambs had any evidence of enteric infections or enteropathy. Acutely ill lambs had azotaemia, haemoconcentration and proteinuria; some lambs had glycosuria or haematuria. Samples of plasma from 22 lambs with nephrosis were compared with similar samples from 82 incontact but asymptomatic lambs. The clinically affected group had significantly elevated plasma urea, creatinine, total protein, globulin, phosphorus and chloride concentrations and significantly reduced plasma calcium concentrations compared with healthy lambs. Affected lambs had a significant reduction also in the calcium:phosphorus ratio. No significant differences between groups was found in plasma concentrations of albumin, glucose, lactate, glycerol, creatine kinase, alkaline phosphatase, sodium, potassium or magnesium.

Seven strains of mice as potential models of bovine pasteurellosis following intranasal challenge with a bovine pneumonic strain of Pasteurella multocida A:3; comparisons of disease and pathological outcomes.

The gram-negative bacterium Pasteurella multocida causes pneumonic and systemic pasteurellosis in bovids for which vaccines are either unavailable or inadequate. The work assessed whether an intranasal P. multocida challenge in mice might provide a model of infection for future vaccine development work. Clinical, pathological and biochemical responses were compared in seven strains of mice challenged with a virulent bovine pneumonic isolate of P. multocida A:3. Six mouse strains (Porton, CD-1, BALB/c, VM, C57BL/10 and C57BL/6) developed clinical signs of pneumonic disease and variable pneumonic lesions 41-70 h post-infection. In contrast, mouse strain RIII became septicaemic within 36 h post-infection. Concentrations of plasma acute phase proteins and serum lipopolysaccharide increased in all mice after infection, and the main or interaction effect of mouse strain and infection status was statistically significant (P<0.05). Responses in C57BL/10 mice showed close similarity to bovine pneumonic and in RIII mice to bovine systemic pasteurellosis.

Molecular epidemiology of Pasteurella multocida in dairy and beef calves.

The molecular epidemiology of Pasteurella multocida has rarely been studied at the farm level in cattle. The aim of this study was to determine whether single or multiple strains of P. multocida tend to exist within farms. Molecular characterisation was carried out on isolates obtained from nasal swabs from 105 calves from 32 randomly selected beef and dairy farms located throughout Scotland, and from 131 calves from 20 farms in the Mayenne region of France, where sampling occurred in response to respiratory disease outbreaks. P. multocida isolates were characterised by random-amplified polymorphic DNA (RAPD) typing and pulsed-field gel electrophoresis (PFGE) using restriction enzyme ApaI. In addition, isolates representative of each farm/RAPD profile combination were typed by multilocus sequence typing (MLST). Among 105 Scottish isolates, 15 RAPD profiles were distinguished. The majority of farms (27/32) had indistinguishable profiles in all positive animals. Five farms had two profiles. Among 140 French isolates, 23 RAPD profiles were distinguished. More within-farm heterogeneity was observed although 10/20 farms had just one profile (E4) in sampled calves. Profile E4 accounted for 60% (84/140) of French isolates. PFGE was more discriminatory than RAPD but confirmed results with respect to within farm homogeneity or heterogeneity of strains, whereas MLST was not discriminatory enough for farm level epidemiology. As in other host species, either several strains or one dominant strain of P. multocida may exist within farms, with evidence for a role of management factors such as movements onto the farm in the number of strains detected.