Comparison of pathophysiologic changes in the lungs of calves challenge exposed with Escherichia coli-derived endotoxin and Pasteurella haemolytica, alone or in combination

Pulmonary responses to intratracheal challenge exposure with Pasteurella haemolytica, with or without Escherichia coli-derived endotoxin, E coli endotoxin alone, or saline solution were compared in anesthetized, mechanically ventilated neonatal calves. Baseline values for dynamic compliance, total pulmonary resistance, functional residual capacity, arterial blood gas tensions, hemogram, leukogram, and systemic and pulmonary arterial pressures were recorded for each calf. After baseline data were obtained, calves were challenge exposed with logarithmic-growth phase P haemolytica organisms with or without E coli endotoxin, E coli endotoxin alone, or saline solution (0.9% NaCl). Physiologic data were obtained immediately after challenge exposure and at various intervals over the next 6 hours. Calves challenge exposed with P haemolytica alone developed sever hypoxemia, had increased alveolar-arterial oxygen difference and threefold increases in total pulmonary resistance, became hypercarbic, had decreased functional residual capacity, and developed systemic hypotension without change in pulmonary arterial pressure. At necropsy, these calves had extensive multifocal areas of necrohemorrhagic and purulent pneumonia. Ratio of extravascular lung water to lung dry weight was not significantly increased in lung specimens obtained from calves challenge exposed with P haemolytica, but ratio of lung wet weight to dry weight was increased, indicating that increased lung wet weight was attributable largely to increased solids and not to fluid alone. (Extravascular lung water measurement excludes fluid from the vascular compartment.) Intratracheal challenge exposure with endotoxin failed to alter lung function and caused minor changes in lung structure consisting of focal areas of hemorrhage and edema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of annexin I and IV and haptoglobin in bronchoalveolar lavage fluid from calves experimentally inoculated with Pasteurella haemolytica

OBJECTIVE: To determine whether annexins or haptoglobin could be detected in bronchoalveolar lavage (BAL) fluid specimens obtained from calves experimentally inoculated with Pasteurella haemolytica. ANIMALS: Twelve 2- to 3-month-old male Holstein calves. PROCEDURE: Pasteurella haemolytica was inoculated into the right lung lobes of each of 6 calves. Six other calves received vehicle alone and were used as control calves. Specimens of BAL fluid were obtained from 3 control and 3 inoculated calves 1 day after inoculation and from the other calves 2 days after inoculation. The amount of annexins I, II, IV, and VI, and haptoglobin in BAL fluid specimens was examined by use of immunoblot analysis. RESULTS: Annexins I and IV were detected in BAL fluid specimens obtained from the right lung lobes of each of the inoculated calves, but annexins II and VI were not. Annexin I also was found in BAL fluid specimens obtained from the left lung lobes of each inoculated calf and from left and right lung lobes of the control calves. By comparison, detection of annexin IV was essentially limited to the right lung lobes of inoculated calves. Haptoglobin was detected in some, but not all, BAL fluid specimens from the right lung lobes of inoculated calves, and its detection in BAL fluid was associated with serum proteins such as albumin. CONCLUSIONS AND CLINICAL RELEVANCE: Annexin IV was detected most specifically in response to inoculation of P haemolytica. This protein could be used as a marker for inflammatory pulmonary disease caused by P haemolytica.     

Detection of annexins I and IV in bronchoalveolar lavage fluids from calves inoculated with bovine herpes virus-1

Annexins are phospholipid-binding proteins and are abundant in the lung. Annexins I and IV, but not II and VI, have been detected in bronchoalveolar lavage (BAL) fluids from calves inoculated with Pasteurella haemolytica, the pathogen for calf pneumonia. In this study, BAL fluids from calves with experimental pneumonia induced by inoculation to right lung lobes of bovine herpes virus-1 (BHV-1), the major viral pathogen for pneumonia, were examined for detection of annexins I and IV. Of 6 calves inoculated with BHV-1, annexins I and IV were coincidentally detected in BAL fluids from right lung lobes of 4 calves, but not in BAL fluids from left lung lobes of 6 inoculated calves or those from left and right lung lobes of 3 control calves. Annexin II and VI were not found in any BAL fluids examined. These results, together with previous findings on calves inoculated with Pasteurella haemolytica, suggest that the release of annexins I and IV onto the alveolar surface is an essential event occurring in response to pulmonary infections of BHIV-1 and Pasteurella haemolytica.     

Importance of neutrophils in the pathogenesis of acute pneumonic pasteurellosis in calves

Acute lung injury was induced in 24 calves by intratracheal inoculation with Pasteurella haemolytica. Calves in groups 1 and 2 were neutrophil depleted, using hydroxyurea given IV. Group 1 calves (n = 7) were inoculated intratracheally with saline solution, and group 2 calves (n = 7) were inoculated with P haemolytica. Group 3 calves (n = 7) had normal numbers of neutrophils and were inoculated with P haemolytica. Group 4 calves (n = 3) were treated acutely with hydroxyurea IV, had normal numbers of neutrophils, and were inoculated with P haemolytica. After inoculation, calves with normal numbers of neutrophils (groups 3 and 4) became hypoxemic 2 hours after inoculation, and hypoxemia persisted until necropsy (6 hours after inoculation). These calves also developed tachypnea, bradycardia, neutropenia, and lymphopenia. Lung lesions consisted of necrosis of the alveolar walls, intra-alveolar hemorrhage, and a severe exudative and necrotizing bronchopneumonia, with accumulation of proteinaceous fluid in alveoli and lymphatics. In neutrophil-depleted calves (groups 1 and 2), blood gas values, heart and respiratory rates, and numbers of circulating leukocytes did not change after inoculation with saline solution or with P haemolytica. At necropsy, the lungs of neutrophil-depleted calves were grossly normal. Therefore, neutrophils were required for the acute lung injury induced by P haemolytica. The protective effect of neutrophil depletion was a specific effect of hydroxyurea because calves with high circulating concentrations of hydroxyurea and calves with normal numbers of neutrophils (group 4) developed lung injury.     

Effects of the synthetic selectin inhibitor TBC1269 on tissue damage during acute Mannheimia haemolytica-induced pneumonia in neonatal calves

OBJECTIVE: To determine effects of the selectin inhibitor TBC1269 on neutrophil-mediated pulmonary damage during acute Mannheimia haemolytica-induced pneumonia in newborn calves. ANIMALS: Eighteen 1- to 3-day-old colostrum-deprived calves. PROCEDURE: Mannheimia haemolytica or saline (0.9% NaCl) solution was inoculated in both cranial lung lobes of 12 and 6 calves, respectively. Calves were euthanatized 2 (saline, n = 3; M haemolytica, n = 4) or 6 hours (saline, n = 3; M haemolytica, n = 8) after inoculation. Four M haemolytica-inoculated calves euthanatized at 6 hours also received TBC1269 (25 mg/kg, IV) 30 minutes before and 2 hours after inoculation. Conjugated diene (CD) concentrations, inducible nitric oxide synthase (iNOS) expression, and apoptotic cell counts were determined in lung specimens collected during necropsy. RESULTS: Conjugated diene concentrations were significantly increased in all M haemolytica-inoculated groups, compared with saline-inoculated groups. Calves treated with TBC1269 had decreased concentrations of CD, compared with untreated calves, although the difference was not significant. Number of apoptotic neutrophils and macrophages increased significantly inTBC1269-treated calves, compared with untreated calves. Inducible nitric oxide synthase was expressed by epithelial cells and leukocytes. However, iNOS was less abundant in airway epithelial cells associated with inflammatory exudates. Degree of iNOS expression was similar between TBC1269-treated and untreated calves. CONCLUSIONS: Mannheimia haemolytica infection in neonatal calves resulted in pulmonary tissue damage and decreased epithelial cell iNOS expression. The selectin inhibitor TCB1269 altered, but did not completely inhibit, neutrophil-mediated pulmonary damage.     

Dexamethasone-induced attenuation of cardiopulmonary dysfunction in endotoxemic calves

Effects of dexamethasone on pulmonary hemodynamics, pulmonary mechanics, and gas exchange were determined in anesthetized (pentobarbital sodium) and paralyzed (pancuronium bromide) calves (9.4 +/- 0.4 weeks old) during 5 hours of endotoxemia. Escherichia coli endotoxin (055-B5) was infused IV at 20 micrograms/kg the 1st hour, followed by a continuous infusion at 10 micrograms/kg/hour for the following 4 hours. Dexamethasone (5 mg/kg) was given IV 18 hours and 1 hour before endotoxin administration, and was also administered IV (1 mg/kg/hr) during endotoxemia. Endotoxin induced large increases in pulmonary artery pressure, pulmonary vascular resistance, alveolar-arterial O2 gradient, alveolar dead-space ventilation, postmortem gravimetric lung weight of bloodless lung, albumin and total protein concentrations in bronchoalveolar lavage fluid, and the number of neutrophils recovered from bronchoalveolar lavage fluid. Endotoxin induced decreases in the cardiac index, dynamic lung compliance, and PaO2. Dexamethasone attenuated most of the cardiopulmonary responses induced by endotoxin, especially during the first 3 hours of endotoxemia. Dexamethasone blocked endotoxin-induced increases in bronchoalveolar lavage albumin, total protein, and neutrophil content. Therefore, glucocorticoids modify endotoxin-induced pulmonary injury in calves, possibly by limiting mobilization of endogenous arachidonic acid.     

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica after intrapulmonary inoculation.

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica were evaluated by measuring immunoglobulin production in blood for 9 days and in pulmonary lavage fluid for 7 days after intrapulmonary inoculation. Clinical signs, pulmonary lesions, pulmonary and systemic inflammatory response, and amount of antigen in lavage fluid were used to evaluate the response of calves to challenge with P haemolytica. The pulmonary response consisted of production of IgG, IgE, and IgM antibodies to P haemolytica antigens and a 17- to 68-fold increase of cells in lavage fluid 8 hours after inoculation, with a gradual decrease toward normal. Antibodies of the IgM isotype to P haemolytica were demonstrated as early as 8 hours through 7 days after inoculation in 3 of 3 calves. Of the anti-P haemolytica isotypes, IgM was found in the highest concentration. In all of the inoculated calves, IgE was found 1 to 2 days after inoculation, and IgG was found in 2 of 3 inoculated calves from day 1 through 7 after inoculation. Detection of IgG correlated with smaller pulmonary lesions. Immunoglobulin A was not detected in lavage fluid. Serum was evaluated for IgG and IgM antibody response to P haemolytica. Specific IgM was detectable 5 days after inoculation, and IgG was detectable 7 days after inoculation. Pasteurella haemolytica antigens were not detected in serum or plasma. A transient increase in neutrophil count was found 8 hours after inoculation, with return to baseline values by 24 hours after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of endotoxemia on lung water and hemodynamics in conscious calves

The effects of endotoxemia on cardiovascular and pulmonary parameters were determined in conscious, 4- to 6-week-old calves. Escherichia coli endotoxin was infused continuously (4 micrograms/kg/hr, IV) for 5 hours. During endotoxemia, pulmonary vascular resistance and mean pulmonary artery pressure increased, and cardiac index, central plasma volume, and mean systemic arterial pressure decreased. Neutrophil, lymphocyte, and platelet counts also decreased. During the first hour of endotoxemia PaO2 decreased, and alveolar-arterial O2 gradient and shunt fraction increased. Lung extravascular thermal volume was increased from 2 to 5 hours. Postmortem extravascular lung water/extravascular dry weight ratio, bronchoalveolar lavage albumin, and poly morphonuclear cell content did not change. Microscopically, the septal capillaries of endotoxemic calves were dilated and engorged with erythrocytes, accompanied by focal accumulations of neutrophils. Intraalveolar edema and hemorrhage were not seen.     

Interactions of cold stress and Pasteurella haemolytica in the pathogenesis of pneumonic pasteurellosis in calves: changes in pulmonary function

Thirteen healthy neonatal Holstein calves were cold stressed twice by hosing with cold water for 20 minutes, 12 hours between hosings. Measurements of the pattern of ventilation [tidal volume (VT), respiratory frequency (f), minute ventilation (VMIN), and functional residual capacity (FRC)], gas exchange properties of the lungs [alveolar ventilation (VA), oxygen uptake (VO2), CO2 production (VCO2), dead space ventilation (VD), dead space/tidal volume ratio (VD/VT), arterial oxygen tension (PaO2), arterial CO2 tension (PaCO2) and alveolar-arterial oxygen difference (AaDO2)] and of the mechanical properties of the pulmonary system [dynamic compliance (Cdyn), pulmonary resistance (RL), and total respiratory system resistance (RRS)] were taken. Calves responded to chilling by increasing VO2 and VCO2 necessitating an increase in VA. This was accomplished by increasing VT with reciprocal decreases in f so that VMIN remained constant. There was no change in Cdyn, RL, or AaDO2. Seven of these 13 calves were then exposed to intratracheal inoculation of 2 X 10(9) organisms of Pasteurella haemolytica, the remaining calves serving as controls. Within 1 hour, calves exposed to P haemolytica had increased VMIN, f, VD/VT, and VD. There was a decrease in PaO2 associated with increased AaDO2, but no change in PaCO2, Cdyn or RL. By 3 hours after inoculation, there were pronounced changes in PaO2 and AaDO2, and Cdyn was reduced below base-line values. By 12 hours after inoculation, calves infected with P haemolytica had increased RL and RRS and PaCO2, in addition to the previously mentioned changes. Data from Pasteurella-exposed calves indicate that gas exchange impairment and peripheral lung injury occur rapidly and that increases in airway resistance develop relatively late in the disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Rabbit pasteurellosis: induced disease and vaccination

Pasteurellosis was induced in rabbits by conjunctival inoculation with 2 strains of Pasteurella multocida. The LD50 of strain P1062 (a bovine isolate) was 10(5.1) colony-forming units and that of strain P1059 (a turkey isolate) was 10(5.5) colony-forming units. Pasteurella-free rabbits were vaccinated IV or mucosally with boiled cells of P multocida or a cross-reactive uridine diphosphogalactose epimerase-deficient mutant of Escherichia coli J5. In rabbits challenge exposed with P multocida strain 1062 or 1059, homologous P multocida strain gave the best protection against fatal bacteremia. Partial protection was provided by J5; mucosal routes of vaccination (aerosol or conjunctival) gave better protection than did the IV route. Serum antibody titers were lower in rabbits vaccinated by mucosal routes than in those vaccinated IV. Cross-reactive IgG and IgM titers to P multocida were demonstrated when rabbits were vaccinated with J5. On the basis of bacteriologic examination of nasal secretions, rabbits that died were considered culture positive sooner than were those that survived. On the basis of bacteriologic examination of blood, rabbits that died were considered culture positive, and those that survived were considered culture negative. Seemingly, heat-stable antigens were protective, the cross-reactive E coli J5 mutant (with only core lipopolysaccharide) provided partial protection against pasteurellosis, and the mucosal route was somewhat useful for cross-protective immunization.     

Effects of endotoxin on lung water, hemodynamics, and gas exchange in anesthetized ponies

Effects of endotoxemia on lung water, hemodynamics, and gas exchange were determined in ponies breathing a mixture of halothane and 100% O2. Escherichia coli endotoxin was infused IV at 20 micrograms/kg of body weight for 1 hour followed by 10 micrograms/kg/hr the subsequent 4 hours. By 0.25 hour, endotoxin increased mean pulmonary artery pressure and pulmonary vascular resistance; this was followed by a return to base-line values by 0.5 and 1 hour, respectively. A 2nd increase in pulmonary vascular resistance occurred by 5 hours of endotoxemia. During the last 2 hours of endotoxin infusion, cardiac index was significantly (P less than 0.05) decreased. Hematocrit was increased from 1 to 5 hours of endotoxemia, whereas, the plasma protein concentration was increased from 2 to 4 hours, indicating a loss of plasma volume. The PaO2 and PaCO2 were unchanged. After 5 hours of endotoxemia, lung extravascular thermal volume, postmortem bronchoalveolar lavage albumin content, and extravascular lung water/extravascular dry weight ratio of bloodless lungs were not increased, indicating no increase in alveolar-capillary permeability or pulmonary edema.     

Pulmonary immunity in calves following stimulation of the gut-associated lymphatic tissue by bacterial exotoxin.

Antibodies in serum and pulmonary lavage fluids were measured in calves following stimulation of the gut-associated lymphatic tissue (GALT) by inoculation of crude leukotoxin of Pasteurella haemolytica into the duodenum through a surgically placed catheter. Nine calves free of P. haemolytica were divided into two groups. Group 1 received an intraduodenal (ID) inoculation of leukotoxin and group 2 received an ID inoculation of phosphate buffered saline. Serum and pulmonary lavage fluids were collected weekly and assayed for antibodies specific to P. haemolytica including immunoglobulin (Ig)G, leukotoxin neutralizing antibodies (LNA), and IgA (lavage fluids only). The multiplicative increase (over baseline) in each class of antibody titer following ID inoculation of leukotoxin, the composite geometric mean increase of all antibodies together, and the composite number of the five antibody titers which increased at least fourfold were computed. Results showed that the geometric mean of each antibody titer and the two composite indices was higher in the GALT-primed groups than in the sham-primed group. The differences were statistically significant (p less than 0.05) for serum IgG and for the two composite indices. This experiment demonstrates for the first time that GALT stimulation by bacterial exotoxins results in increased pulmonary antibody levels in calves.     

Induction of pulmonary antibodies to Pasteurella haemolytica following intraduodenal stimulation of the gut-associated lymphatic tissue in cattle.

The induction of pulmonary antibodies to a bacterial antigen following intraduodenal (D) stimulation of the gut-associated lymphatic tissue (GALT) was investigated. Six calves were divided into two groups of three calves each. The GALT-primed calves received an ID dose of live Pasteurella haemolytica A1 followed by a subcutaneous (SC) dose of killed P. haemolytica. The sham-primed calves received an ID dose of phosphate-buffered saline solution (PBSS) followed by a SC dose of killed bacteria. Serum and pulmonary lavage fluids were collected weekly from each calf and assayed for titers of leukotoxin neutralizing antibodies (LNA), as well as IgG and IgA (lavage fluids only) to P. haemolytica. The GALT-primed calves responded to the ID stimulation by bacteria with increased serum IgG. The sham-primed calves had no change in antibody titers following ID stimulation. The GALT-primed calves had increased serum IgG, lavage IgG and IgA and increased LNA titers in both lavage fluids and serum following the SC dose of killed bacteria. The sham-primed calves demonstrated only an increase in serum IgG following the SC inoculation. A challenge study to evaluate if antibodies induced by GALT stimulation could reduce pulmonary lesions was performed using six calves divided into two groups. One group received an ID dose of P. haemolytica followed two weeks later by a SC dose of killed P. haemolytica. The sham vaccinated calves received an ID dose of PBSS followed in two weeks by a SC dose of killed bacterin. Calves were challenged by an intrapulmonary dose of live P. haemolytica A1 eleven days after the SC inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary lesions induced by Pasteurella haemolytica in neutrophil sufficient and neutrophil deficient calves.

The role of neutrophils in the development of peracute lung lesions of bovine pneumonic pasteurellosis was investigated. Eight calves were divided into two groups of four calves each. Group I was treated with intravenous phosphate-buffered saline and served as the neutrophil sufficient calves. Group II was treated with intravenous hydroxyurea which produced a state of neutropenia. When peripheral blood neutrophil numbers dropped below 300 cells/microL in group II, all calves were challenged with an intrabronchial bolus of Pasteurella haemolytica in the log phase of growth. An acute inflammatory process occurred in both groups of calves indicated by a rise in body temperature. While pulmonary lesions occurred in both groups by six hours postinoculation, they varied in pathological characteristics. Pulmonary lesions in the neutrophil sufficient calves consisted of fibrinopurulent alveolitis-bronchiolitis with associated alveolar septal necrosis, interlobular edema, and intravascular thrombi. The neutrophil deficient calves had extensive intra-alveolar edema, interlobular edema, intraalveolar hemorrhage, atelectasis, and focal areas of alveolar septal necrosis. These results show that P. haemolytica can induce severe pulmonary tissue damage through both neutrophil dependent and neutrophil independent mechanisms.     

Sequential titration of bovine lung and serum antibodies after parenteral or pulmonary inoculation with Pasteurella haemolytica

Calves were vaccinated by intrabronchial or subcutaneous injection of formalinized Pasteurella haemolytica. Antibody in serum, nasal washings, and bronchoalveolar washings was titrated sequentially before and after calves were vaccinated and then challenge exposed with live homologous bacteria. Bronchoalveolar washings were collected by fiberoptics bronchoscopy, and antibody was titrated by indirect (antiglobulin) bacterial agglutination. Responsiveness to vaccination was related in initial serum antibody concentrations. Calves with serum antibody titers of 1:20 or more were nonresponsive, whereas with few exceptions, calves having titers of less than 1:20 responded to vaccination. Results indicated that serum and lung antibody were induced by subcutaneous or by intrabronchial inoculation of formalinized P haemolytica. By either route of immunization, serum antibody was more persistent than was lung antibody, and pulmonary challenge exposure with live P haemolytica did not alter existing titers.     

Experimental production of bovine respiratory tract disease with bovine viral diarrhea virus

Five 6-month-old calves were inoculated with bovine viral diarrhea (BVD) virus (n = 3) or Pasteurella haemolytica (n = 2) endobronchially with a fiberoptic bronchoscope. Five additional calves were inoculated sequentially with BVD virus followed by P haemolytica at a 5-day interval. Blood samples were collected daily from the calves for bacterial isolation. Clinical signs of respiratory tract disease in calves were recorded daily. If the calves survived, they were killed for necropsy 3 or 4 days after inoculation with P haemolytica (or 8 days after inoculation with BVD virus). The extent and nature of pulmonary lesions in the calves were determined, and the lower portion of the respiratory tract (lungs and trachea) was examined for both these organisms. The 3 calves, inoculated with BVD virus only, developed mild clinical signs mainly manifested as fever, nasal discharge, and occasional cough. Approximately 2% to 7% of the total lung capacity of these calves was pneumonic. Mild clinical signs and localized lesions involving about 15% of the lung volume developed in the 2 calves exposed to P haemolytica only. However, severe fibrinopurulent bronchopneumonia and pleuritis involving 40% to 75% of lung volume developed in the 5 calves inoculated sequentially with BVD virus and P haemolytica. The possible role BVD virus may have in bovine respiratory tract disease is discussed.     

Pasteurella haemolytica leukotoxin induces histamine release from bovine pulmonary mast cells.

Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to induce histamine release from bovine pulmonary mast cells isolated by enzymatic dispersion of lung tissue. Histamine was measured by a radioimmunoassay technique. Leukotoxic culture supernatant of P. haemolytica significantly released histamine in a time and concentration-related manner. This effect was lost when culture supernatant was heat-inactivated or preincubated with leukotoxin neutralizing rabbit serum. Preincubation of the mast cells with propranolol or p-bromophenacyl bromide reduced the histamine-releasing effect of leukotoxin, while verapamil enhanced release. Experimental infection of calves with P. haemolytica A1 reduced the total histamine content of pulmonary mast cells recovered at postmortem. Histamine release induced by P. haemolytica leukotoxin is likely an important factor in the pathogenesis of bovine pneumonic pasteurellosis.     

Treatment of experimentally induced pneumonic pasteurellosis of young calves with tilmicosin.

Twenty four (24) healthy male Holstein calves (< 70 kg) were each experimentally infected by intrabronchial inoculation of 4.0 x 10(9) viable cells of Pasteurella haemolytica-AI (B122) at Time = 0 h. At 1 h following inoculation animals received either: 1) Sham treatment with sterile 0.85% saline SC (n = 12); or 2) a single injection of 10 mg tilmicosin per kg body weight (n = 12). Calves that were non-infected and tilmicosin-treated were also included for determining tilmicosin concentrations in serum and lung tissue at 1, 2, 4, 6, 8, 24, 48, and 72 h (n = 3-per time). In the infected calves, response to therapy was monitored clinically. Serum samples were collected for determination of tilmicosin concentrations using HPLC. Any animal becoming seriously ill was humanely killed. Complete necropsy examinations were performed on all animals and included gross pathologic changes, bacteriologic analysis, histopathology, and determination of pulmonary concentrations of tilmicosin. Tilmicosin treated animals responded significantly better to therapy than saline-treated control calves. Clinical assessment of calves during the study indicated that tilmicosin-treated calves had significantly improved by T = 8 h compared to satine-treated animals (P < 0.05). At necropsy tilmicosin-treated calves had significantly less severe gross and histological lesions (P < 0.05) of the pulmonary tissue. Of the 12 saline-treated calves, 92% (11/12) had Pasteurella haemolytica-A1 in lung tissue, while of the tilmicosin-treated calves 0% (0/12) cultured positive for P. haemolytica. Mean (+/- standard error) serum tilmicosin concentrations in infected calves peaked at 1 h post-injection (1.10 +/- 0.06 micrograms/mL) and rapidly decreased to 0.20 +/- 0.03 microgram/mL, well below the MIC of 0.50 microgram/mL for P. haemolytica-A1 (B122), by 12 h. These serum concentrations were very similar to serum concentrations of tilmicosin in non-infected tilmicosin-treated calves. Lung tissue concentrations of the antibiotic were comparatively high, even at 72 h post-infection (6.50 +/- 0.75 ppm). Lung tissue concentrations at 72 h were significantly higher in experimentally infected calves than in non-infected tilmicosin-treated animals (P < 0.05). These data demonstrate that tilmicosin was effective in treating experimentally-induced pneumonic pasteurellosis as determined by alleviation of clinical signs, pathological findings at post mortem, and presence of viable bacteria from the lung. Concentrations substantially above MIC for P. haemolytica were present in lung tissue even at 72 h following a single subcutaneous injection of 10 mg tilmicosin per kg body weight.     

Escherichia coli O157:H7 in the gallbladders of experimentally infected calves.

Fifteen weaned calves (age 89-141 days) were treated with dexamethasone (0.25 mg/kg, IV) for 3 days before, the day of, and the day after inoculation with 10 colony-forming units of either Escherichia coli O157:H7 (strain 86-24, which produces Shiga toxin 2 and intimin; n = 13) or nonpathogenic E. coli (strain 123, which does not produce Shiga toxin or intimin; n = 2). All calves were necropsied 4 days after inoculation. Histologic lesions of attaching and effacing bacteria were observed in the large intestine (12/13) and in the gallbladder mucosa (5/13) of calves inoculated with E. coli 86-24. Cholecystitis was present in 12 of 13 calves that received E. coli 86-24. Inoculum bacteria were recovered from the distal colons or feces (13/13) and gallbladders (3/4) of calves inoculated with 86-24.