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 10⁹ 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 10⁹ 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 1 h 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.     

Altered endothelium-dependent α-adrenergic vascular reactivity following exposure toPasteurella haemolytica antigens

Rats were injected with sterile saline (controls), 10⁵ cfu ofPasteurella haemolytica (biotype A) obtained from a commercial vaccine, or a commercialPasteurella leukotoxoid vaccine. Three days after vaccination, the animals were killed and the thoracic aorta was removed. In some experiments the vascular endothelium was mechanically removed. Each isolated aorta was placed in a tissue bath and the biophysical responses to methoxamine (-1 agonist) were determined. In separate experiments the endothelial surface was examined by scanning electron microscopy. In endothelium-intact vessels both vaccines appeared to enhance the contractile response to methoxamine. On the other hand, in endothelial-denuded vessels, the methoxamine-mediated contractile response was enhanced in theP. haemolytica-treated group but not in animals vaccinated with leukotoxoid. Furthermore, scanning electron microscopy revealed deposition of a fibrin-like material on the endothelial surface of vaccinated animals. These results suggest that exposure to vaccine-derivedP. haemolytica antigens alters the morphology and adrenergic responsiveness of vascular smooth muscle.Abbreviations BPP bovine pneumonic pasteurellosis - cfu colony forming units - EC₅₀ concentration giving 50% of the maximum response - IP intraperitoneal - mN millinewtons - pD₂ -log(EC₅₀)     

Mouse model of haemorrhagic septicaemia: dissemination and multiplication of Pasteurella multocida B:2 in vital organs after intranasal and subcutaneous challenge in mice

Haemorrhagic septicaemia (HS) is an endemic disease of bovines, occuring in most tropical regions of Asia and Africa. In the present study, the suitability of using mice to study pathogenesis of HS was assessed using mortality, mean death time and bacterial multiplication in vital organs after infection with live P multocida. Mice were infected with 10⁵, 10³ and 10¹?cfu of P. multocida B:2 via intranasal and subcutaneous routes along with control groups. Bacterial multiplication in lung, liver and spleen of mice were determined at 24 h interval after intranasal and subcutaneous challenge. More than 80 % of challenged mice died within 48 h of inoculation, irrespective of the dose and route of inoculation. A heavy bacterial load (up to 10⁸?cfu) was observed in lung, liver and spleen of mice titrated at 24 h and following death of mice. Results of the present study indicate that even ten bacteria are enough to cause mortality in mice and the organism multiplies rapidly in respiratory epithelium and disseminated to other vital organs viz liver and spleen suggesting the important role of mouse model in investigating the pathogenesis and challenge studies during vaccine development.     

A Quantitative Flurometric Assay for the Measurement of Antibody to Pasteurella haemolytica in Cattle

A rapid, simple fluorometric method is described for measuring antibody to Pasteurella haemolytica in sera of cattle. Various antigen preparations were compared for the test including live, formalin-killed and phenol-killed P. haemolytica. A preparation composed of formalin-killed organisms from a 22 hour culture gave consistent results and was used in the studies. The test was reproduciable with percent coefficients of variation for fluorescent signal unit values on ten or more replicate samples ranging from 5.7 to 28.0. Sera from calves vaccinated by aerosol exposure to live P. haemolytica had up to a five-fold increase in antibody titer as measured by the flurometric method test during a 21 day period. Fluorometric method titers were comparable to those obtained by the indirect bacterial agglutination test. There was no seroconversion to P. haemolytica in calves vaccinated by aerosol exposure of P. multocida. The major advantages of the fluorometric method test over conventional methods are that the assay does not require serial dilutions of serum samples and thus limits time and effort to determine antibody titers.     

Protective effect following intranasal exposure of goats to live Pasteurella multocida B:2

This study aimed to determine the effect of intranasal exposure to low doses of Pasteurella multocida B:2 on survival of goats challenged with high doses of the same organism. Eighteen goats were selected and divided into three groups. Goats of group 1 were exposed intranasally twice, with a two-week interval, to 7× 10⁶ cfu/ml of live P. multocida B:2. Goats of group 2 were not exposed to P. multocida B:2 but were kept together with the exposed group 1. Goats of group 3 remained as unexposed controls and were kept separated from the other two groups. Serum samples were collected at weekly intervals to determine the antibody levels. At week 5 post exposure, all goats were challenged subcutaneously with 3.7× 10¹⁰ cfu/ml of live P. multocida B:2. Following challenge exposure, 8 (67%) goats (4 goats from each of groups 1 and 2) were killed owing to haemorrhagic septicaemia. Four goats were killed peracutely within 48 h post challenge, while the other four goats were killed acutely between 2 and 4 days post challenge. None of the goats of group 3 were killed for haemorrhagic septicaemia. Goats of groups 1 and 2 showed significantly (p<0.05) higher antibody levels following the first intranasal exposure to P. multocida B:2. However, only group 1 retained the significantly (p<0.05) high antibody levels following a second intranasal exposure, and remained significantly (p<0.05) higher than groups 2 and 3 at the time of challenge. P. multocida B:2 was successfully isolated from various organs of goats that were killed between 1 and 4 days post challenge.     

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.     

Efficacy of an inactivated recombinant vaccine encoding a fimbrial protein of Pasteurella multocida B:2 against hemorrhagic septicemia in goats

This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the fimbrial protein of Pasteurella multocida B:2 following intranasal vaccination against hemorrhagic septicemia in goats. Goats were vaccinated intranasal with 10⁶ CFU/mL of the recombinant vaccine (vaccinated group) and 10⁶ CFU/mL of pET32/LIC vector without fimbrial protein (control group). All three groups were kept separated before all goats in the three groups were challenged with 10⁹ CFU/mL of live pathogenic P. multocida B:2. During the course of study, both serum and lung lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay. It was found that goats immunized with the inactivated recombinant vaccine developed a strong and significantly (p < 0.05) higher specific IgA and IgG responses in both serum and lung lavage fluid samples compared to the control and unvaccinated groups. Following intratracheal challenge, the rate of isolation was 17% for the vaccinated group, 67% for the control group and 100% for the unvaccinated group. However, none of the goat from the vaccinated group had P. multocida B:2 in the liver, tonsil and heart. Therefore, the study revealed that an inactivated recombinant vaccine significantly provides significant protection against high dose challenge and enhances the stimulation of the local and systemic immunities.     

Pharmacokinetic–pharmacodynamic integration and modelling of oxytetracycline for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3 hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration–time curve (AUC_0–∞)/MIC ratios of 30.0 and 24.3 hr for M. haemolytica and P. multocida, respectively. Corresponding AUC_0–∞/MIC ratios based on MICs in broth were 656 and 745 hr, respectively. PK‐PD modelling of in vitro bacterial time–kill curves for oxytetracycline in serum established mean AUC_0–24 hr/MIC ratios for 3log₁₀ decrease in bacterial count of 27.5 hr (M. haemolytica) and 60.9 hr (P. multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48‐hr periods were 197 mg/kg (M. haemolytica) and 314 mg/kg (P. multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25‐ and 27‐fold lower (7.8 and 11.5 mg/kg) for M. haemolytica and P. multocida, respectively.     

Oat cells in the pathology of ovine pneumonia-pleurisy

Light microscope observations on oat cells in the ovine pneumonia-pleurisy complex are presented. This study is based on the experimental production of the disease by viruses and Pasteurella haemolytica. Oat cells appeared only in necrotic lesions associated with large numbers of P. haemolytica in thi pneumonic lung. It is suggested that oat cells originate from blood monocytes, which transform into the oat shape when developing in the necrotic, hypoxic environment created by P. haemolytica. They were not, however, observed to be phagocytic. Oat cells are characteristic of pneumonic pasteurellosis but are not pathognomonic because they can also be found in extrapulmonary locations and in other pathological conditions of the lungs.     

Dominant expression of interleukin-8 vs interleukin-1β and tumour necrosis factor alpha in lungs of lambs experimentally infected with Mannheimia haemolytica

Abstract

AIMS: To quantify the number of cells infected with Mannheimia haemolytica and expressing interleukin (IL)-1β, tumour necrosis factor alpha (TNFα) and IL-8 using immunohistochemistry, and to measure the immunoreactivity of cytokines in pulmonary tissue extracts using ELISA, in the lung of lambs experimentally infected with M. haemolytica, and to compare the patterns of expression of cytokines in airways at different times post-infection (p.i.).

METHODS: Twenty 3-month-old lambs of both sexes were randomly assigned to two groups, viz infected (n=15), and uninfected controls (n=5). Each lamb in the infected group was inoculated with 1.5 x 10⁹ cfu M. haemolytica in 5 mL sterile nutrient broth, control lambs were inoculated with 5 mL sterile nutrient broth and clinical signs were monitored. Infected and control animals were killed at 1, 3, 5, 7, and 15 days p.i. Histopathology and immunohistochemistry were conducted to determine the number of immunolabelled cells in pneumonic lungs, and study the pattern of expression of IL-1β, TNFα and IL-8 in lung extracts using ELISA.

RESULTS: Lesions in bronchi and bronchioles ranged from epithelial desquamation to bronchiolitis obliterans and necrosis. The alveoli had areas of seroproteinaceous fluid, fibrin and bacterial aggregates that evolved to foci of pyogranulomatous inflammation with clustered inflammatory cells, referred to as ‘oat cells’. M. haemolytica antigen was observed in the cytoplasm of inflammatory cells. Labelling of IL-1β, TNFα and IL-8 was observed in bronchial and bronchiolar epithelial cells, alveolar exudate, and in interstitial inflammatory infiltrate, with increased expression on 1 and 3 days p.i. for IL-1β and TNFα, and 1, 3, and 5 days p.i. for IL-8. In lung tissue extracts, peak concentrations of IL-1β (55 (SD 5) ng/mL), TNFα (92 (SD 6) pg/mL) and IL-8 (8 [SD 2] μg/mL) occurred at 3 days p.i.

CONCLUSIONS: The results of this study suggested that the inflammatory cytokines IL-1β, TNFα and IL-8 may play an important role in enhancing the biological response to M. haemolytica, and contribute to the development of lesions in the lung in pulmonary pasteurellosis in sheep. Given that the expression of IL-8 in lung was much greater than that of IL-1β and TNFα, anti-cytokine agents directed at this mediator could be useful in the prevention and treatment of this disease.     

Mucosal immunization provides better protection than subcutaneous immunization against <Emphasis Type="Italic">Pasteurella multocida</Emphasis> (B:2) in mice preimmunized with the outer membrane proteins

To investigate the effect of boosting immunity via mucosal route vis-a-vis parenteral route in the mouse model of haemorrhagic septicaemia, mice preimmunized with OMP of Pasteurella multocida (B:2) were immunized with 10² cfu of P. multocida via intranasal and subcutaneous routes. Mice were challenged through intranasal route (natural route of infection) with 10⁸ cfu 14 days after immunization. Group of mice which were immunized intranasally showed significant protection (P < 0.05) of 88% as compared to 50% protection in group of mice immunized subcutaneously. In the control group of mice, 100% mortality occurred within 48 h. of challenge. The results of present study indicated that boosting of immunity via mucosal route in mice preimmunized with OMP provided better protection against P. multocida. This study may have implications for developing better vaccination strategies for the natural host.     

Pharmacokinetic/pharmacodynamic integration and modelling of amoxicillin for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time‐kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time‐kill data, based on the sigmoidal E_max equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log₁₀ reduction) and 4log₁₀ reduction in bacterial counts. For these levels, mean AUC_(0–24 h)/MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (C_max)/MIC ratios of 13.9:1 and 25.2:1, area under concentration–time curve (AUC_0–∞)/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).     

Pharmacokinetic and pharmacodynamic integration and modelling of marbofloxacin in calves for Mannheimia haemolytica and Pasteurella multocida

The pharmacokinetics (PK) and pharmacodynamics (PD) of marbofloxacin were established in calves for six strains of each of the pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. The distribution of marbofloxacin into inflamed (exudate) and non-inflamed (transudate) tissue cage fluids allowed comparison with the serum concentration–time profile. To establish the PD profile, minimum inhibitory concentration (MIC) was determined in Mueller–Hinton broth (MHB) and calf serum.Moderately higher MICs were obtained for serum compared to MHB. An initial integration of PK–PD data established C_max/MIC ratios of 45.0 and AUC_24h/MIC values of 174.7 h, based on serum MICs, for both bacterial species. Using bacterial time-kill curves, generated ex vivo for serum marbofloxacin concentrations, PK–PD modelling established three levels of growth inhibition: AUC_24h/MIC ratios for no reduction, 3 log₁₀ and 4 log₁₀ reductions in bacterial count from the initial inoculum count were 41.9, 59.5 and 68.0 h for M. haemolytica and 48.6, 64.9 and 74.8 h for P. multocida, on average respectively. Inter-strain variability for 3 log₁₀ and 4 log₁₀ reductions in bacterial count was smaller for P. multocida than for M. haemolytica. In conjunction with literature data on MIC₉₀ values, the present results allowed prediction of dosages for efficacy for each organism for the three levels of growth inhibition.     

The Effect of Pasteurella haemolytica A2 Infection on Phagocytosis Efficiency of Caprine Broncho‐Alveolar Macrophages

An experiment was designed to study the in vivo effect of Pasteurella haemolytica A2 infection on the phagocytosis activity of caprine broncho‐alveolar macrophages and the extent of pneumonic lesions. Twelve healthy local Kacang goats, about 7 months of age, were divided into two groups of six. Goats in group 1 were inoculated intratracheally with 4 ml inoculum containing 2.8 × 10⁹ colony‐forming units (CFU)/ml of Staphylococcus aureus. Goats in group 2 were inoculated intratracheally with 4 ml of inoculum containing 9.5 × 10⁸ CFU/ml of Pasteurella haemolytica A2 isolated earlier from pneumonic lungs of goat. At intervals of 3 and 7 days post‐challenge five goats from each group were killed and the lungs were washed with sterile phosphate‐buffered saline. Smears were prepared from the lung washing fluid and the number of macrophages with phagocytic activity was determined. At day 3 post‐infection, goats of both groups showed a similar pattern of pneumonic lesion. The lung washing fluid of goats in group 2 was found to contain numerous neutrophils and macrophages. Goats in group 2 showed significantly (P < 0.05) higher extent of lung lesions than group 1. Similarly, the average extent of lung lesions was significantly (P < 0.05) more severe in group 2 at day 7 post‐infection. The lung washing fluid contained mostly macrophages. The phagocytic activity following S. aureus infection was more efficient and significantly (P < 0.01) higher compared with infection by P. haemolytica A2. There were weak correlations between the extent of pneumonic lesion and the phagocytic activity. Thus, goats with poor phagocytic activity were likely to develop more extensive lung lesions.     

Serum antibodies to Pasteurella haemolytica lipopolysaccharide: relationship to experimental bovine pneumonic pasteurellosis

An enzyme-linked immunosorbent assay was used to determine the serum antibody response to Pasteurella haemolytica lipopolysaccharide (LPS) for calves vaccinated with saline solution, a formalin-killed P haemolytica bacterin, or live P haemolytica. Bacterin-vaccinated calves had a lower antibody response to LPS than did calves vaccinated with live P haemolytica. Calves vaccinated with either saline solution or the bacterin were more susceptible to intrapulmonic challenge exposure with P haemolytica than were calves vaccinated with liver organisms. Serum antibody responses to P haemolytica LPS did not seem important for resistance to challenge exposure, because there was no significant correlation (P greater than 0.05) between the lung lesion score and antibody response to P haemolytica LPS. There was a highly significant correlation (P less than 0.001) between antibody detected against P haemolytica LPS and that against formalin-killed P haemolytica. Competitive binding studies indicated that P haemolytica LPS is a major antigenic determinant on the surface of P haemolytica. There did not seem to be substantial cross-reaction between LPS from P haemolytica and that from Escherichia coli (serotype O26:B6).     

Pharmacokinetic–pharmacodynamic integration and modelling of florfenicol in calves

Florfenicol was administered subcutaneously to 10 calves at a dose of 40 mg/kg. Pharmacokinetic–pharmacodynamic (PK‐PD) integration and modelling of the data were undertaken using a tissue cage model, which allowed comparison of microbial growth inhibition profiles in three fluids, serum, exudate and transudate. Terminal half‐lives were relatively long, so that florfenicol concentrations were well maintained in all three fluids. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined in vitro for six strains each of the calf pneumonia pathogens, Mannhemia haemolytica and Pasteurella multocida. An PK‐PD integration for three serum indices provided mean values for P. multocida and M. haemolytica, respectively, of 12.6 and 10.4 for C_max/MIC, 183 and 152 h for AUC_0–24 h/MIC and 78 and 76 h for T>MIC. Average florfenicol concentrations in serum exceeded 4 × MIC and 1.5 × MIC for the periods 0–24 and 48–72 h, respectively. Ex vivo growth inhibition curves for M. haemolytica and P. multocida demonstrated a rapid (with 8 h of exposure) and marked (6 log₁₀ reduction in bacterial count or greater) killing response, suggesting a concentration‐dependent killing action. During 24‐h incubation periods, inhibition of growth to a bacteriostatic level or greater was maintained in serum samples collected up to 96 h and in transudate and exudate samples harvested up to 120 h. Based on the sigmoidal E_max relationship, PK‐PD modelling of the ex vivo time–kill data provided AUC_0–24 h/MIC serum values for three levels of growth inhibition, bacteriostatic, bactericidal and 4 log₁₀ decrease in bacterial count; mean values were, respectively, 8.2, 26.6 and 39.0 h for M. haemolytica and 7.6, 18.1 and 25.0 h for P. multocida. Similar values were obtained for transudate and exudate. Based on pharmacokinetic and PK‐PD modelled data obtained in this study and scientific literature values for MIC distributions, Monte Carlo simulations over 100 000 trials were undertaken to predict once daily dosages of florfenicol required to provide 50% and 90% target attainment rates for three levels of growth inhibition, namely, bacteriostasis, bactericidal action and 4 log₁₀ reduction in bacterial count.     

Antimicrobial resistance in bovine respiratory disease: Auction market- and ranch-raised calves

This study compared changes in prevalence and antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni in feedlot calves derived from the auction market (AUCT; n = 299) and from a single-ranch source (RANCH; n = 300). In the AUCT calves, the prevalence of Mannheimia haemolytica decreased, whereas Histophilus somni increased over the feeding period. The AUCT calves showed an increase in isolates not susceptible to tulathromycin for all bovine respiratory disease (BRD) pathogens, an increase in Pasteurella multocida and Histophilus somni isolates not susceptible to oxytetracycline, and an increase in Pasteurella multocida isolates not susceptible to florfenicol. In the RANCH calves, the prevalence of all 3 BRD pathogens was high at feedlot entry and decreased significantly during the study period. In RANCH calves, there was a significant increase in Pasteurella multocida isolates not susceptible to oxytetracycline, tulathromycin, and florfenicol. Surprisingly, there was a significant decrease in Mannheimia haemolytica isolates that were not susceptible to oxytetracycline, tilmicosin, and tulathromycin.     

Identification of <Emphasis Type="BoldItalic">Pasteurella multocida</Emphasis> capsular types isolated from rabbits and other domestic animals in Mexico with respiratory diseases

Pasteurella multocida is the causative agent of pasteurellosis, a major disease in most domestic animals and livestock. In this study, a total of 34 isolates of P. multocida from rabbits and other domestic animals from Mexico with respiratory diseases underwent polymerase chain reaction-based capsular typing. One sheep isolate was found to belong to capsular serogroup D, whereas the rest of the rabbit, sheep, cattle, pig, goat, and duck isolates belonged to capsular serogroup A of P. multocida. This is the first report of capsular type A in P. multocida isolates from rabbits and duck origin in Mexico.     

Pharmacokinetics and pharmacodynamics of gamithromycin in pulmonary epithelial lining fluid in naturally occurring bovine respiratory disease in multisource commingled feedlot cattle

The objectives of this study were to determine (i) whether an association exists between individual pharmacokinetic parameters and treatment outcome when feeder cattle were diagnosed with bovine respiratory disease (BRD) and treated with gamithromycin (Zactran^®) at the label dose and (ii) whether there was a stronger association between treatment outcome and gamithromycin concentration in plasma or in the pulmonary epithelial lining fluid (PELF) effect compartment. The study design was a prospective, blinded, randomized clinical trial utilizing three groups of 60 (362–592 lb) steers/bulls randomly allocated within origin to sham injection or gamithromycin mass medication. Cattle were evaluated daily for signs of BRD by a veterinarian blinded to treatment. Animals meeting the BRD case definition were enrolled and allocated to a sample collection scheme consisting of samples for bacterial isolation (bronchoalveolar lavage fluid and nasopharyngeal swabs) and gamithromycin concentration determination (PELF and plasma). Gamithromycin susceptibility of M. haemolytica (n = 287) and P. multocida (n = 257) were determined using broth microdilution with frozen panels containing gamithromycin at concentrations from 0.03 to 16 μg/mL. A two‐compartment plasma pharmacokinetic model with an additional compartment for gamithromycin in PELF was developed using rich data sets from published and unpublished studies. The sparse data from our study were then fit to this model using nonlinear mixed effects modeling to estimate individual parameter values. The resulting parameter estimates were used to simulate full time–concentration profiles for each animal in this study. These profiles were analyzed using noncompartmental methods so that PK/PD indices (AUC₂₄/MIC, AUC_∞/MIC, C_MAX/MIC) could be calculated for plasma and PELF (also T>MIC) for each individual. The calculated PK/PD indices were indicative that for both M. haemolytica and P. multocida a higher drug exposure in terms of concentration, and duration of exposure relative to the MIC of the target pathogen, was favorable to a successful case outcome. A significant association was found between treatment success and PELF AUC_0–24/MIC for P. multocida. The calves in this study demonstrated an increased clearance and volume of distribution in plasma as compared to the healthy calves in two previously published reports. Ultimately, the findings from this study indicate that higher PK/PD indices were predictive of positive treatment outcomes.     

Immunologic study and optimization of Salmonella delivery strains expressing adhesin and toxin antigens for protection against progressive atrophic rhinitis in a murine model

Mice were intranasally inoculated at various times to optimize the vaccination strategy with a new live candidate vaccine expressing the antigens CP39, FimA, PtfA, and ToxA of Pasteurella multocida and F1P2 of Bordetella bronchiseptica in an attenuated live Salmonella system to protect against progressive atrophic rhinitis (PAR). Sixty BALB/c mice were divided equally into 4 groups. The group A mice were vaccinated only at 12 wk of age, the group B mice received a primary vaccination at 9 wk of age and a booster at 12 wk of age, the group C mice received a primary vaccination at 6 wk of age and boosters at 9 and 12 wk of age, and the group D mice were inoculated intranasally with sterile phosphate-buffered saline as a control. The humoral and mucosal immune responses of groups A, B, and C increased significantly compared with those of the control group. Expression of the cytokines interleukin-4 and interferon-γ in splenocytes also increased significantly. In addition, the group B mice exhibited significantly fewer gross lesions in lung tissue compared with the other vaccinated groups after challenge with a virulent P. multocida strain. These results indicate that a strategy of double intranasal vaccination can optimize protection against PAR.