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1.  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.
PMCID: PMC1189402  PMID: 9242998
2.  The frequency, distribution and effects of antibodies, to seven putative respiratory pathogens, on respiratory disease and weight gain in feedlot calves in Ontario. 
During 1983-85, 279 calves requiring treatment for bovine respiratory disease and 290 comparison (control) animals from 15 different groups of feedlot calves were bled on arrival and again at 28 days postarrival. Their sera were then analyzed for antibodies to seven putative respiratory pathogens. On arrival, the prevalences of indirect agglutination titers to Pasteurella haemolytica, P. haemolytica cytotoxin, Mycoplasma bovis and M. dispar were greater than 50%, the prevalence of titers to bovine virus diarrhea virus (BVDV) was approximately 40%, and the prevalences of titers to infectious bovine rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) were all below 25%. Seroconversion during the first month after arrival occurred in more than half the calves to P. haemolytica cytotoxin, PIV3 and RSV. Seroconversion of agglutination titers to P. haemolytica, Mycoplasma and BVDV occurred in about 40% of calves, and seroconversion to IBRV was infrequent (less than 5%). Initial titers were negatively correlated to subsequent titer changes within organism. Initial titers, and titer changes between organisms were essentially independent. Light calves had an increased risk of being selected for treatment for respiratory disease. Seroconversion to P. haemolytica cytotoxin, RSV and BVDV were predictive of respiratory disease cases, explaining approximately 69% of all respiratory disease cases in the feedlots. It was not possible to accurately predict weight gain or relapse from the serological data.
PMCID: PMC1255725  PMID: 2766158
3.  The pulmonary clearance of Pasteurella haemolytica in calves given Corynebacterium parvum and infected with parainfluenza-3 virus. 
Four control calves were aerosolized with parainfluenza-3 and one week later with Pasteurella haemolytica. Three calves were given Corynebacterium parvum at a dose of 15 mg/m2 body surface area, infected with parainfluenza-3 virus one week later, and aerosolized with P. haemolytica two weeks after C. parvum injection. All calves were killed four hours after P. haemolytica exposure and the bacterial retention in the lung was determined. Parainfluenza-3 viral infection did not exert any suppressive effect on pulmonary clearance of P. haemolytica in six out of seven calves used. However, the bacterial colony counts in the lungs of control calves were higher (P less than 0.05) than those in calves given C. parvum. Hence, C. parvum appeared to enhance bacterial clearance. Despite the marked influx of neutrophils into the lungs after the bacterial inoculation, the neutrophil:macrophage ratio in lavage samples was less in calves given C. parvum than in the control calves. The alveolar macrophages in C. parvum treated calves were generally larger but did not differ significantly (P less than 0.05) from those in the controls. There was no significant (P less than 0.05) correlation between the percentages of alveolar macrophages and the bacterial clearance. In calves given C. parvum, bacterial clearance was enhanced in those calves which had larger macrophages.
PMCID: PMC1320202  PMID: 6280824
4.  Vaccination studies against experimental bovine Pasteurella pneumonia. 
Vaccination-challenge experiments were conducted in colostrum-deprived calves to evaluate the efficacy of Pasteurella bacterins and vaccines against experimental pneumonic pasteurellosis. Calves were vaccinated with formalin-killed bacterins and live vaccines, then challenge exposed intratracheally with P. haemolytica or P. multocida. Infectious bovine rhinotracheitis virus was inoculated intranasally three to four days prior to P. haemolytica challenge-exposure. All calves were examined for macroscopic and microscopic lesions after being found dead or following euthanasia four to seven days after challenge exposure with the bacterial pathogen. Clinical, hematological, and pathological responses to challenge exposure in aluminum hydroxide absorbed P. haemolytica and P. multocida bacterin-treated calves were consistent with the pneumonic lesions of pulmonary pasteurellosis in the control calves. An oil-adjuvanted P. haemolytica bacterin limited clinical and pathological responses in the affected calves whereas a P. multocida oil-adjuvanted bacterin did not. Both clinical and pathological responses to challenge exposure in calves vaccinated with live Pasteurella vaccines were less severe than those of the control calves. Vaccine effectiveness appeared to be dose dependent.
PMCID: PMC1255304  PMID: 3300921
5.  Transmission of Bovine viral diarrhea virus 1b to susceptible and vaccinated calves by exposure to persistently infected calves 
Bovine viral diarrhea virus (BVDV) persistently infected (PI) calves represent significant sources of infection to susceptible cattle. The objectives of this study were to determine if PI calves transmitted infection to vaccinated and unvaccinated calves, to determine if BVDV vaccine strains could be differentiated from the PI field strains by subtyping molecular techniques, and if there were different rates of recovery from peripheral blood leukocytes (PBL) versus serums for acutely infected calves. Calves PI with BVDV1b were placed in pens with nonvaccinated and vaccinated calves for 35 d. Peripheral blood leukocytes, serums, and nasal swabs were collected for viral isolation and serology. In addition, transmission of Bovine herpes virus 1 (BHV-1), Parainfluenza-3 virus (PI-3V), and Bovine respiratory syncytial virus (BRSV) was monitored during the 35 d observation period.
Bovine viral diarrhea virus subtype 1b was transmitted to both vaccinated and nonvaccinated calves, including BVDV1b seronegative and seropositive calves, after exposure to PI calves. There was evidence of transmission by viral isolation from PBL, nasal swabs, or both, and seroconversions to BVDV1b. For the unvaccinated calves, 83.2% seroconverted to BVDV1b. The high level of transmission by PI calves is illustrated by seroconversion rates of nonvaccinated calves in individual pens: 70% to 100% seroconversion to the BVDV1b. Bovine viral diarrhea virus was isolated from 45 out of 202 calves in this study. These included BVDV1b in ranch and order buyer (OB) calves, plus BVDV strains identified as vaccinal strains that were in modified live virus (MLV) vaccines given to half the OB calves 3 d prior to the study. The BVDV1b isolates in exposed calves were detected between collection days 7 and 21 after exposure to PI calves. Bovine viral diarrhea virus was recovered more frequently from PBL than serum in acutely infected calves. Bovine viral diarrhea virus was also isolated from the lungs of 2 of 7 calves that were dying with pulmonary lesions. Two of the calves dying with pneumonic lesions in the study had been BVDV1b viremic prior to death. Bovine viral diarrhea virus 1b was isolated from both calves that received the killed or MLV vaccines. There were cytopathic (CP) strains isolated from MLV vaccinated calves during the same time frame as the BVDV1b isolations. These viruses were typed by polymerase chain reaction (PCR) and genetic sequencing, and most CP were confirmed as vaccinal origin. A BVDV2 NCP strain was found in only 1 OB calf, on multiple collections, and the calf seroconverted to BVDV2. This virus was not identical to the BVDV2 CP 296 vaccine strain. The use of subtyping is required to differentiate vaccinal strains from the field strains. This study detected 2 different vaccine strains, the BVDV1b in PI calves and infected contact calves, and a heterologous BVDV2 subtype brought in as an acutely infected calf. The MLV vaccination, with BVDV1a and BVDV2 components, administered 3 d prior to exposure to PI calves did not protect 100% against BVDV1b viremias or nasal shedding. There were other agents associated with the bovine respiratory disease signs and lesions in this study including Mannheimia haemolytica, Mycoplasma spp., PI-3V, BRSV, and BHV-1.
PMCID: PMC1176294  PMID: 16187545
6.  Pasteurella haemolytica lipopolysaccharide-associated protein induces pulmonary inflammation after bronchoscopic deposition in calves and sheep. 
Infection and Immunity  1995;63(9):3595-3599.
The lipopolysaccharide (LPS)-associated protein (LAP) was extracted from Pasteurella haemolytica serotype A1 strains L101 (bovine origin) and 82-25 (ovine origin). Extracts contained 0.017% total LPS and appeared as only two bands at 14 and 16.6 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To determine the extent of pulmonary inflammation induced by LAP and its possible role in the pathogenesis of pneumonic pasteurellosis, LAP (500 micrograms in pyrogen-free saline [PFS]) was deposited by fiber-optic bronchoscopy into the dorsum of the caudal portion of the cranial lobe of the right lung of calves (strain L101 LAP) and sheep (strain 82-25 LAP). LPS (500 micrograms in PFS), 3-h P. haemolytica cultures (1.6 x 10(8) to 1.9 x 10(8) CFU in PFS), and PFS alone were deposited similarly as controls. At necropsy, 24 h after deposition, gross and histologic pulmonary lesions of calves and sheep given LAP, LPS, and P. haemolytica were similar and consisted of various degrees of acute bronchopneumonia (relative severities of lesions induced: LAP < LPS < live organisms). By subjective histologic interpretation and semiquantitative morphometry, animals given LAP had the highest percentage of macrophages per alveolar lumen and the lowest percentage of neutrophils. The lesions from animals given LPS were more severe than those given LAP, but the morphometric cell counts were similar. In contrast, animals inoculated with P. haemolytica had lesions typical of this agent, consisting of many neutrophils, proteinaceous exudate, and a few macrophages. Morphometrically, these lesions had the highest numbers of neutrophils and the lowest numbers of macrophages. These studies show that LAP can induce an inflammatory response in the alveolar lumens and may play a role in the pathogenesis of pneumonic pasteurellosis.
PMCID: PMC173499  PMID: 7642296
7.  Pathogens of Bovine Respiratory Disease in North American Feedlots Conferring Multidrug Resistance via Integrative Conjugative Elements 
Journal of Clinical Microbiology  2014;52(2):438-448.
In this study, we determined the prevalence of bovine respiratory disease (BRD)-associated viral and bacterial pathogens in cattle and characterized the genetic profiles, antimicrobial susceptibilities, and nature of antimicrobial resistance determinants in collected bacteria. Nasopharyngeal swab and lung tissue samples from 68 BRD mortalities in Alberta, Canada (n = 42), Texas (n = 6), and Nebraska (n = 20) were screened using PCR for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus, bovine herpesvirus 1, parainfluenza type 3 virus, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Excepting bovine herpesvirus 1, all agents were detected. M. haemolytica (91%) and BVDV (69%) were the most prevalent, with cooccurrence in 63% of the cattle. Isolates of M. haemolytica (n = 55), P. multocida (n = 8), and H. somni (n = 10) from lungs were also collected. Among M. haemolytica isolates, a clonal subpopulation (n = 8) was obtained from a Nebraskan feedlot. All three bacterial pathogens exhibited a high rate of antimicrobial resistance, with 45% exhibiting resistance to three or more antimicrobials. M. haemolytica (n = 18), P. multocida (n = 3), and H. somni (n = 3) from Texas and Nebraska possessed integrative conjugative elements (ICE) that conferred resistance for up to seven different antimicrobial classes. ICE were shown to be transferred via conjugation from P. multocida to Escherichia coli and from M. haemolytica and H. somni to P. multocida. ICE-mediated multidrug-resistant profiles of bacterial BRD pathogens could be a major detriment to many of the therapeutic antimicrobial strategies currently used to control BRD.
PMCID: PMC3911356  PMID: 24478472
8.  Bovine viral diarrhea viral infections in feeder calves with respiratory disease: interactions with Pasteurella spp., parainfluenza-3 virus, and bovine respiratory syncytial virus. 
The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in a group of stocker calves suffering from acute respiratory disease. The calves were assembled after purchase from Tennessee auctions and transported to western Texas. Of the 120 calves, 105 (87.5%) were treated for respiratory disease. Sixteen calves died during the study (13.3%). The calves received a modified live virus BHV-1 vaccine on day 0 of the study. During the study, approximately 5 wk in duration, sera from the cattle, collected at weekly intervals, were tested for BVDV by cell culture. Sera were also tested for neutralizing antibodies to BVDV types 1 and 2, bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV). The lungs from the 16 calves that died during the study were collected and examined by histopathology, and lung homogenates were inoculated onto cell cultures for virus isolation. There were no calves persistently infected with BVDV detected in the study, as no animals were viremic on day 0, nor were any animals viremic at the 2 subsequent serum collections. There were, however, 4 animals with BVDV type 1 noncytopathic (NCP) strains in the sera from subsequent collections. Viruses were isolated from 9 lungs: 7 with PI-3V, 1 with NCP BVDV type 1, and 1 with both BVHV-1 and BVDV. The predominant bacterial species isolated from these lungs was Pasteurella haemolytica serotype 1. There was serologic evidence of infection with BVDV types 1 and 2, PI-3V, and BRSV, as noted by seroconversion (> or = 4-fold rise in antibody titer) in day 0 to day 34 samples collected from the 104 survivors: 40/104 (38.5%) to BVDV type 1; 29/104 (27.9%) to BVDV type 2; 71/104 (68.3%) to PI-3V; and 81/104 (77.9%) to BRSV. In several cases, the BVDV type 2 antibody titers may have been due to crossreacting BVDV type 1 antibodies; however, in 7 calves the BVDV type 2 antibodies were higher, indicating BVDV type 2 infection. At the outset of the study, the 120 calves were at risk (susceptible to viral infections) on day 0 because they were seronegative to the viruses: 98/120 (81.7%), < 1:4 to BVDV type 1; 104/120 (86.7%) < 1:4 to BVDV type 2; 86/120 (71.7%) < 1:4 to PI-3V; 87/120 (72.5%) < 1:4 to BRSV; and 111/120 (92.5%) < 1:10 to BHV-1. The results of this study indicate that BVDV types 1 and 2 are involved in acute respiratory disease of calves with pneumonic pasteurellosis. The BVDV may be detected by virus isolation from sera and/or lung tissues and by serology. The BVDV infections occurred in conjunction with infections by other viruses associated with respiratory disease, namely, PI-3V and BRSV. These other viruses may occur singly or in combination with each other. Also, the study indicates that purchased calves may be highly susceptible, after weaning, to infections by BHV-1, BVDV types 1 and 2, PI-3V, and BRSV early in the marketing channel.
PMCID: PMC1189606  PMID: 10935880
9.  Induction of immunity against pneumonic pasteurellosis following experimental infection in calves. 
Immunity against pneumonic pasteurellosis was studied in calves after recovery from experimental respiratory disease with Pasteurella haemolytica. Nine calves were exposed to aerosols of parainfluenza-3 virus and Pasteurella haemolytica A1 six days apart to produce respiratory disease. After recovery from the disease, these nine principal and four control calves were challenged with aerosols of bovine herpesvirus 1 and P. haemolytica A1 four days apart. With this viral-bacterial challenge, the nine principal animals failed to develop clinical responses to this bacterial challenge and their lungs did not show the growth of P. haemolytica on cultures, whereas two of four control calves had elevated temperatures and developed necropurulent pneumonia with the isolation of P. haemolytica from the lungs. The principal calves had developed high levels of cytotoxin neutralizing antibodies in their sera following parainfluenza-3 virus-P. haemolytica infection. This demonstrated that immunity against pneumonic pasteurellosis can be achieved, with a suggestion that further search for an effective vaccine for P. haemolytica is warranted.
PMCID: PMC1255154  PMID: 3017526
10.  A group level analysis of the associations between antibodies to seven putative pathogens and respiratory disease and weight gain in Ontario feedlot calves. 
The associations, at the group level, between serological titer to Pasteurella haemolytica surface antigens (Ph), Pasteurella haemolytica cytotoxin (Ph-cytox), infectious bovine rhinotracheitis virus (IBRV), bovine virus diarrhea virus (BVDV), parainfluenza-3 virus (PIV3), respiratory syncytial virus (RSV), Mycoplasma dispar (Md), M. bovis (Mb), and respiratory disease treatment rates, relapse rates, and 28 day weight gains were investigated in 14 groups of calves entering two feedlots during years 1983-1985, in Ontario. Based on least squares regression analyses, seroconversion rates to Mb and BVDV were predictive of increased respiratory disease rates, and seroconversion rates to Ph, Ph-cytox, Md and PIV3 were predictive of decreased weight gains. The R2 for predicting weight gains was much higher than for morbidity rates (0.75 vs 0.47 respectively). Titer data were not predictive of relapse rates. Group level analyses were performed because calves are managed as groups (e.g. pens) in commercial feedlots. Only BVDV seroconversion rates were related to increased risk of respiratory disease at both the individual and group levels of organization. Mycoplasma may be important factors in causing respiratory disease, and their relationship to potentiating the effects of other respiratory pathogens needs further investigation.
PMCID: PMC1255666  PMID: 2165846
11.  Influence of β2-Integrin Adhesion Molecule Expression and Pulmonary Infection with Pasteurella haemolytica on Cytokine Gene Expression in Cattle 
Infection and Immunity  2000;68(7):4274-4281.
β2-Integrins are leukocyte adhesion molecules composed of alpha (CD11a, -b, -c, or -d) and beta (CD18) subunit heterodimers. Genetic CD18 deficiency results in impaired neutrophil egress into tissues that varies between conducting airways and alveoli of the lung. In this study, we investigated whether CD18 deficiency in cattle affects proinflammatory cytokine (PIC) expression in pulmonary tissue after respiratory infection with Pasteurella haemolytica. Cattle were infected with P. haemolytica via fiberoptic deposition of organisms into the posterior part of the right cranial lung lobe. Animals were euthanized at 2 or 4 h postinoculation (p.i.), and tissues were collected to assess PIC gene expression using antisense RNA probes specific for bovine interleukin-1α (IL-1α), IL-1β, IL-6, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) along with the β-actin (β-Act) housekeeping gene. Expression of PIC was induced at 2 h p.i. in P. haemolytica-infected cattle and continued to 4 h p.i. At 2 h p.i., induction of gene expression and increase of cells that expressed PIC were observed both in CD18+ and CD18− cattle after inoculation of P. haemolytica. The induction of gene expression with P. haemolytica inoculation was more prominent in CD18− cattle than in CD18+ cattle by comparison to pyrogen-free saline (PFS)-inoculated control animals. At 4 h p.i., however, the induction of PIC, especially IL-1α, IL-6, and IFN-γ, in the lungs of CD18+ cattle inoculated with P. haemolytica was greater than that in lungs of the CD18− cattle. IFN-γ and TNF-α genes were not increased in P. haemolytica-inoculated CD18− cattle lungs compared to the PFS-inoculated control lungs at 4 h p.i. In PFS-inoculated lungs, we generally observed a higher percentage of cells and higher level of gene expression in the lungs of CD18− cattle than in the lungs of CD18+ cattle, especially at 4 h p.i. The rate of neutrophil infiltration into the lungs of CD18− cattle at 2 h p.i. was significantly higher than that of CD18+ cattle; at 4 h p.i., there was no difference between the two groups. These data suggest that β2-integrins may contribute to the induction of expression of some PIC genes, as a consequence of P. haemolytica infection.
PMCID: PMC101743  PMID: 10858245
12.  A guinea pig model of bovine pneumonic pasteurellosis. 
The induction of pneumonic pasteurellosis in guinea pigs (Cavia porcellus) was examined. Specific pathogen free male guinea pigs were anesthetized and a tracheostomy performed to introduce 10(5), 10(4) or 10(3) Pasteurella haemolytica-A1 into the left principal bronchus. The surgical site was closed with tissue adhesive and staples and the animals were monitored for signs of respiratory tract infection. Within 24 hours after inoculation they became depressed, anorectic, pyretic and dyspneic. Fibrinous pleuropneumonia with prominent areas of necrosis and hemorrhage was present. Pericardial effusion was a frequent finding. There was infiltration of the pleura and alveoli with degenerate heterophils and macrophages, a hyperplastic mesothelium and fibrin exudation on the pleura and within alveoli. Hemorrhage, congestion, consolidation, edema and fibrin exudation were prominent in the hilar region of the lungs. Bacterial colonies were evident in all airways. More bacteria were recovered from infected lungs than were inoculated (p less than 0.05) indicating P. haemolytica was actively multiplying in the lungs. Hematological and clinical chemistry data were consistent with fibrinous pneumonia, however, blood cultures were positive for P. haemolytica in 61% (11/18) of animals sampled. Examination of pneumonic pasteurellosis in guinea pigs may be useful in studying pathogenetic and pathological features applicable to bovine pneumonic pasteurellosis (shipping fever pneumonia).
PMCID: PMC1255618  PMID: 2306663
13.  Epidemiological study of enzootic pneumonia in dairy calves in Saskatchewan. 
A field study involving 325 calves from 17 dairy herds in Saskatchewan was conducted to determine the risk of enzootic pneumonia and to assess its association with a number of factors. Two different case definitions of pneumonia were used in the analyses: the first was based on producers' treatment risk (CASE1) and the second was based on semimonthly clinical examinations of calves by the research veterinarian (CASE2). The risk of pneumonia based on CASE1 was 39% and on CASE2 was 29%. The measure of agreement between CASE1 and CASE2 at the calf level of analysis was poor (kappa = 0.24, SE = 0.02) and at the herd level of analysis was moderate (kappa = 0.40, SE = 0.12). The mortality risk from pneumonia was 1.8% and a variety of infectious organisms were isolated from pneumonic lungs. Twenty-seven percent of the calves had inadequate (total IgG < or = 800 mg/dL) levels of passively acquired antibodies as measured by radial immunodiffusion. The proportion of seropositive titers in calves within the first two weeks of age was 94% to parainfluenza 3 virus (PI3V) and bovine respiratory syncytial virus (BRSV), 73% to Pasteurella haemolytica (Ph), 68% to bovine viral diarrhea virus (BVDV), 67% to infectious bovine rhinotracheitis virus (IBRV), 46% to Mycoplasma dispar (Md), 44% to Haemophilus somnus (Hs), and 21% to Mycoplasma bovis (Mb). At the calf level of analysis and after adjusting for clustering, there was a negative association (p = 0.10) between the diagnosis of pneumonia based on CASE2 and total IgG levels and Ph titers (rPh).(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263636  PMID: 8269363
14.  Exposure of calves to aerosols of parainfluenza-3 virus and Pasteurella haemolytica. 
The present study was undertaken to investigate whether sequential exposure to aerosols of parainfluenza-3 virus followed by Pasteurella haemolytica, or P. haemolytica followed by parainfluenza-3 virus, could lead to the production of pulmonary lesions in conventionally-raised calves. Twenty male calves with low serum antibody titres to both organisms were placed in five equal groups. Synergism of parainfluenza-3 virus and P. haemolytica was not demonstrated in any of the sequentially infected groups and pulmonary lesions were mild in all challenged calves. Clinical signs of disease were not present after exposure to parainfluenza-3 virus although the virus was repeatedly isolated from nasal secretions of all inoculated calves. Exposure to P. haemolytica produced a transient response which consisted of increased rectal temperatures and respiratory rates, with a mild neutrophilic leukocytosis and a mild left shift present six hours postinoculation and returning to normal within 24 hours. Results from this study suggest, although do not confirm, that reduced pulmonary clearance of inhaled P. haemolytica in parainfluenza-3 virus infected calves does not necessarily lead to production of severe pulmonary lesions and that previous exposure to aerosols of P. haemolytica may not enhance secondary parainfluenza-3 virus infection.
PMCID: PMC1235969  PMID: 6320999
15.  Serological titers to bovine herpesvirus 1, bovine viral diarrhea virus, parainfluenza 3 virus, bovine respiratory syncytial virus and Pasteurella haemolytica in feedlot calves with respiratory disease: associations with bacteriological and pulmonary cytological variables. 
Acute and convalescent serum samples were taken from 59 calves with signs of respiratory disease (cases) and 60 clinically normal animals (controls) during their first month in the feedlot. Sera were analyzed for antibodies to bovine parainfluenza 3 (PI3) virus by hemagglutination inhibition, to bovine viral diarrhea (BVD) virus, bovine respiratory syncytial (BRS) virus and bovine herpesvirus 1 (BHV1) by virus neutralization, and to Pasteurella haemolytica by indirect agglutination (PhIA) and cytotoxin neutralization (PhCN) tests. There was minimal evidence of serological activity to BHV1. Serological activity to the other agents occurred commonly and the prevalence of acute titers and their mean values was similar in case and control groups. Mean convalescent PI3 and P. haemolytica (PhIA) titers were higher in controls than cases (p < 0.01) but, otherwise, convalescent titers did not differ between groups. The incidence of seroconversion was similar in both groups for all agents except for PI3 virus which was more frequent in controls than cases (p < 0.0001). There was a positive association between PhIA and CN seroconversion and isolation of P. haemolytica from bronchoalveolar lavage (BAL) fluid (p < 0.1). The measure of agreement (kappa) between seroconversion with the P. haemolytica PhIA and PhCN tests was 0.51. Bacteriological and cytological evaluations of the respiratory tract were made using BAL. No associations were evident between serological titers and pulmonary cytology. A multivariate logistic analysis was used to evaluate associations between disease status and serological, bacteriological and cytological data. Cases were positively associated with the presence of neutrophils and Pasteurella multocida in BAL fluid and negatively associated with PI3 virus and PhIA seroconversion.
PMCID: PMC1263557  PMID: 1335831
16.  Induction of CD18-Mediated Passage of Neutrophils by Pasteurella haemolytica in Pulmonary Bronchi and Bronchioles 
Infection and Immunity  1999;67(2):659-663.
Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs during P. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (β2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected with P. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P < 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.
PMCID: PMC96369  PMID: 9916073
17.  Pulmonary recruitment of neutrophils and bacterial clearance in mice inoculated with aerosols of Pasteurella haemolytica or Staphylococcus aureus. 
Pulmonary alveolar macrophages are considered to be the main phagocytic cell of the pulmonary defense mechanism. However recent studies indicate that neutrophils may also participate in the defense against inhaled bacteria. The aim of this investigation was to study in mice the correlation between numbers of phagocytic cells in the bronchoalveolar space and the pulmonary clearance of bacteria. White mice were exposed to aerosols of Pasteurella haemolytica (n = 129) or Staphylococcus aureus (n = 129) in three different experimental replicates. Another group of mice (n = 22) was sham exposed to an aerosol of sterile phosphate buffered solution in a single replicate. Animals were sacrificed at various times postaerosolization. The numbers of neutrophils and alveolar macrophages in lung lavages and the pulmonary bacterial clearance rates were determined and statistically analysed. No significant differences (p greater than 0.05) were observed in the rates of pulmonary clearance between the two genera of bacteria, but P. haemolytica had a significant (p less than 0.05) replicate effect. The number of alveolar macrophages was not significantly affected by either bacteria or phosphate buffered solution. Exposure to P. haemolytica resulted in dramatic, significant (p less than 0.01) but transient increases in neutrophils in the bronchoalveolar space as well as a significant (p less than 0.01) increase in the weights of lung. The correlation between neutrophils and clearance was positive for P. haemolytica but negative for S. aureus. These results indicate that both species of bacteria are rapidly eliminated from the lung despite a rather different cellular response.
PMCID: PMC1236181  PMID: 4041978
18.  Preliminary studies with a live streptomycin-dependent Pasteurella multocida and Pasteurella haemolytica vaccine for the prevention of bovine pneumonic pasteurellosis. 
Twelve Pasteurella-free Holstein-Friesian calves were used in a study to test the efficacy of a live streptomycin-dependent Pasteurella multocida A:3 and streptomycin-dependent Pasteurella haemolytica A1 vaccine. The calves were inoculated intramuscularly twice at 14-day intervals with either the streptomycin-dependent vaccine, containing 1 X 10(6) colony forming units/mL P. multocida and 4 X 10(8) colony forming units/mL P. haemolytica, commercial bacterin, or phosphate buffered saline. Two weeks following the second vaccination, all calves were challenged by intranasal inoculation of 10(8) TCID50/4.0 mL infectious bovine rhinotracheitis virus followed three days later by intratracheal injection with 2.3 X 10(7) colony forming units/mL of a 16 hour culture of P. multocida A:3 and 2.6 X 10(8) colony forming units/mL of an 8 hour culture of P. haemolytica A1. Seven days after challenge with Pasteurella, calves were killed for collection of tissues at necropsy. Each calf was given a score based on macroscopic and microscopic lesions. The scores for the calves receiving live vaccines were significantly lower (p less than 0.025) than those for the controls. Also, the calves receiving live vaccines had a significant (p less than 0.05) increase in the level of serum antibody to P. haemolytica. The results of this preliminary study showed that the streptomycin-dependent vaccine offered better protection than the commercial bacterin against a virulent homologous challenge.
PMCID: PMC1236194  PMID: 3907804
19.  Seroepidemiology of undifferentiated fever in feedlot calves in western Canada. 
The relationships between 4 bacterial and 3 viral antibody titers and morbidity (undifferentiated fever (UF)) and mortality were investigated in recently weaned beef calves. Blood samples from 100 animals that required treatment for UF (Cases) and 100 healthy control animals (Controls) were obtained: upon arrival at the feedlot (Arrival), at the time of selection as a Case or Control (Selection), and at approximately 33 d of the feeding period (Convalescent). Seroconversion to Pasteurella haemolytica antileukotoxin was associated with an increased risk of UF (OR = 2.83); however, seroconversion to bovine herpesvirus-1 G-IV glycoprotein was associated with a decreased risk of UF (OR = 0.43). Higher Arrival bovine viral diarrhea virus antibody titer was associated with a decreased risk of UF (OR = 0.83). Increases in Mycoplasma alkalescens antibody titer after Arrival were associated with an increased risk of UF (OR = 1.10). Higher Arrival Haemophilus somnus antibody titer and increases in Haemophilus somnus antibody titer after Arrival were both associated with a decreased risk of UF (OR = 0.76 and OR = 0.78). The odds of overall mortality (OR = 5.09) and hemophilosis mortality (OR = 11.31) in Cases were significantly (P < 0.05) higher than in the Controls. Higher Arrival bovine herpesvirus-1 antibody titer was associated with an increased risk of mortality (OR = 1.30). Protective immunity to Pasteurella haemolytica antileukotoxin, Haemophilus somnus, bovine herpesvirus-1 G-IV glycoprotein, bovine viral diarrhea virus, and Mycoplasma spp. may be necessary to reduce the occurrence of UF. Animals with UF are at an increased risk of overall and hemophilosis mortality.
PMCID: PMC1539654  PMID: 9919366
20.  Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin. 
Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis.
PMCID: PMC1236028  PMID: 6372970
21.  Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease 
The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in 2 groups of stocker calves with acute respiratory disease. Both studies used calves assembled after purchase from auction markets by an order buyer and transported to feedyards, where they were held for approximately 30 d. In 1 study, the calves were mixed with fresh ranch calves from a single ranch. During the studies, at day 0 and at weekly intervals, blood was collected for viral antibody testing and virus isolation from peripheral blood leukocytes (PBLs), and nasal swabs were taken for virus isolation. Samples from sick calves were also collected. Serum was tested for antibodies to bovine herpesvirus-1 (BHV-1), BVDV1a, 1b, and 2, parainfluenza 3 virus (PI3V), and bovine respiratory syncytial virus (BRSV). The lungs from the calves that died during the studies were examined histopathologically, and viral and bacterial isolation was performed on lung homogenates. BVDV was isolated from calves in both studies; the predominant biotype was noncytopathic (NCP). Differential polymerase chain reaction (PCR) and nucleic acid sequencing showed the predominant subtype to be BVDV1b in both studies. In 1999, NCP BVDV1b was detected in numerous samples over time from 1 persistently infected calf; the calf did not seroconvert to BVDV1a or BVDV2. In both studies, BVDV was isolated from the serum, PBLs, and nasal swabs of the calves, and in the 1999 study, it was isolated from lung tissue at necropsy. BVDV was demonstrated serologically and by virus isolation to be a contributing factor in respiratory disease. It was isolated more frequently from sick calves than healthy calves, by both pen and total number of calves. BVDV1a and BVDV2 seroconversions were related to sickness in selected pens and total number of calves. In the 1999 study, BVDV-infected calves were treated longer than noninfected calves (5.643 vs 4.639 d; P = 0.0902). There was a limited number of BVDV1a isolates and, with BVDV1b used in the virus neutralization test for antibodies in seroconverting calves' serum, BVDV1b titers were higher than BVDV1a titers. This study indicates that BVDV1 strains are involved in acute respiratory disease of calves with pneumonic Mannheimia haemolytica and Pasteurella multocida disease. The BVDV2 antibodies may be due to cross-reactions, as typing of the BVDV strains revealed BVDV1b or 1a but not BVDV2. The BVDV1b subtype has considerable implications, as, with 1 exception, all vaccines licensed in the United States contain BVDV1a, a strain with different antigenic properties. BVDV1b potentially could infect BVDV1a-vaccinated calves.
PMCID: PMC227002  PMID: 12146890
22.  Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida 
Applied and Environmental Microbiology  2012;78(18):6683-6688.
Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.
PMCID: PMC3426682  PMID: 22798357
23.  The immunohistochemical detection of Mycoplasma bovis and bovine viral diarrhea virus in tissues of feedlot cattle with chronic, unresponsive respiratory disease and/or arthritis. 
The Canadian Veterinary Journal  2001;42(11):857-860.
The purpose of this study was to determine the frequency of selected pathogens in the tissues of a group of feedlot cattle with chronic disease (most often respiratory disease and/or arthritis). Samples of lung and joint tissues from 49 feedlot animals that had failed to respond to antibiotic therapy were tested by immunohistochemical staining for the antigens of Mycoplasma bovis, Haemophilus somnus, Pasteurella (Mannheimia) hemolytica, and bovine viral diarrhea virus (BVDV). Mycoplasma bovis was demonstrated in over 80% of cases, including in 45% of joints and 71% of lungs tested. Mycoplasma bovis was the only bacterial pathogen identified in the joints. Haemophilus somnus and Pasteurella (Mannheimia) haemolytica were found in 14% and 23% of cases, respectively, and were confined to the lungs in all instances. Infection with BVDV was demonstrated in over 40% of cases. Mycoplasma bovis and BVDV were the most common pathogens persisting in the tissues of these animals that had failed to respond to antibiotic therapy.
PMCID: PMC1476660  PMID: 11708203
24.  Aerosol vaccination of calves with pasteurella haemolytica against experimental respiratory disease. 
Three experiments were conducted on calves in which the efficacy of vaccination with live Pasteurella haemolytica in aerosol was tested by challenge with sequential aerosol exposure to bovine herpesvirus 1 and P. haemolytica. Neither single nor multiple aerosol vaccinations protected against the experimental disease. Macroscopically recognizable rhinitis, tonsillitis, tracheitis and pneumonia occurred in both controls and vaccinates. In one experiment as many as three aerosol vaccinations with live P. haemolytica for up to 20 minutes failed to elicit clinical signs in exposed calves. Pasteurella haemolytica was isolated less frequently from tissues of vaccinated calves than from those of nonvaccinated calves. Pasteurella haemolytica was isolated from deep nasal swabs of 4/14 vaccinated calves five and six days after viral exposure. It was concluded that although bovine herpesvirus 1 vaccination has been shown previously to prevent the experimental disease produced by bovine herpesvirus 1-P. haemolytica, live P. haemolytica vaccination by aerosol will not provide the same protection.
PMCID: PMC1320326  PMID: 6290014
25.  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.
PMCID: PMC1263651  PMID: 7511483

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