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–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.     

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.     

Pasteurella multocida and its role in porcine pneumonia

Pasteurella multocida has been recognized as a contributor to debilitating and fatal porcine pneumonia for at least 120 years and there continues to be sustained, unabated high prevalence of the organism in cases submitted for diagnostic work up. Understanding of its role in disease has been limited, in part because of difficulty in reproducing the disease experimentally with capsular type A strains of P. multocida, the predominant type associated with porcine pneumonia. This limitation has stymied the development of improved methods for disease control. In this review, the reports of efforts to reproduce the disease are compared. Reports have indicated induction of pneumonia in combined infections with agents such as hog cholera virus, pseudorabies virus and Mycoplasma hyopneumoniae. Pneumonia has been induced with intratracheal or endobronchial inoculation of anesthetized swine using capsular type A strains. Substantial recent progress in understanding the putative virulence attributes and molecular genetics of P. multocida will likely lead to better understanding of the host-parasite and parasite-parasite interactions in porcine pneumonia associated with this organism. In particular, it seems important to consider the role of biofilm formation in the pathogenesis of this disease. Ultimately, this understanding should provide a foundation for better methods for induction of the experimental disease, development of improved diagnostics, development of better therapeutic/prophylactic pharmaceutical approaches and development of immunoprophylactic products.     

猪的胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

在已经建立猪胸膜肺炎放线杆菌(APP)、猪多杀性巴氏杆菌(PM)、副猪嗜血杆菌(HPS)的单项PCR诊断方法的基础上,通过对扩增条件的优化,成功地建立了APP、PM、HPS的复合PCR实验室诊断方法,利用一次PCR反应,即可同时扩增出APP的342bp、PM的457bp和HPS的821bp的特异性片段。该复合PCR能同时检测到100个每种细菌或50pg的APP或HPS的DNA和500pg的PM的DNA。该三重复合PCR的敏感性同已报道的单PCR一致。该方法的建立对临床上进行这3种疾病的鉴别诊断和混合感染的检测都具有重要意义。     

副猪嗜血杆菌、多杀性巴氏杆菌和胸膜肺炎放线杆菌多重PCR检测方法的建立及应用

在已建立的副猪嗜血杆菌(HPS)、多杀性巴氏杆菌(Pm)、胸膜肺炎放线杆菌(APP)的单重PCR检测方法的基础上,通过对扩增条件的优化,利用一次PCR反应可同时扩增出APP的256 bp、Pm的457 bp、HPS的822 bp的特异性片段,建立了HPS、Pm、APP的多重PCR实验室诊断方法。该复合PCR可检测60 pg的HPS、120 pg的Pm、50 pg的APP。利用该方法检测猪繁殖与呼吸综合征病毒(PRRSV)阳性猪体内分离到的39株细菌,结果显示HPS、Pm、APP分别为12、16、2株,对39份病料的检测与常规细菌分离鉴定结果一致,可见该方法适用于HPS、APP、Pm的临床快速检测。     

猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。     

猪胸膜肺炎放线杆菌、肺炎支原体和多杀性巴氏杆菌联合检测芯片的研制及应用

本研究旨在建立联合检测胸膜肺炎放线杆菌、猪肺炎支原体和多杀性巴氏杆菌的DNA芯片.用7个从3种病菌基因组中扩增出的不同特异性靶DNA制作基因芯片,并对芯片的靶DNA和探针浓度、杂交温度、重复性、特异性和灵敏度进行了研究.结果表明,检测芯片的特异性强,能与测试的李氏放线杆菌、猪鼻支原体和副猪嗜血杆菌等9种病原区分;灵敏度高,在50μL标记反应体系中,能检测到10～50 pg基组DNA,芯片可重复利用.用芯片对44株目标菌的不同型标准菌株、分离株和疫苗株进行了检测.其信号值≥1 000,信号噪音比(SNR)≥6.用芯片对45头病猪和97头健康猪的临床样品选择培养物进行了检测,其检出率分别为多杀性巴氏杆菌71.1%和49.5%、胸膜肺炎放线杆菌42.2%和26.8%、猪肺炎支原体20%和22.7%,混合感染率分别为42.2%和24.7%.在检测临床样品时,芯片法与PCR的符合率为97.8%～100%,与分离鉴定法的符合率为87.6%～95.6%.研究表明,研制的芯片特异性强、敏感性高、可重复使用,是一种能有效用于胸膜肺炎放线杆菌、猪多杀性巴氏杆菌和猪肺炎支原体鉴定和联合检测的新工具.     

Pharmacokinetic/pharmacodynamic integration of cefquinome against Pasteurella Multocida in a piglet tissue cage model

To explore the in vivo antimicrobial activity of cefquinome against Pasteurella multocida in piglets, a piglet tissue cage infection model was used in this study. After the population of P. multocida reached 10⁷ CFU/mL in a tissue cage, piglets received an intramuscular administration of cefquinome at 0.2, 0.4, 0.8, 1, 2, and 4 mg/kg once daily for 3 days. To assess the tissue cage pharmacokinetics (PKTCF) of cefquinome, tissue cage fluid was collected for cefquinome analysis at 1, 3, 6, 9, 12, and 24 hr after each of the 3 daily drug administrations. Bacteria were counted every 24 hr after drug administration and at 48 and 72 hr after the last administration. Evaluation of the relationship between pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect showed that the surrogate of %T > minimum inhibitory concentration (MIC) (R² = 0.981) was the best PK/PD index that correlated with effectiveness of cefquinome against P. multocida. The respective values of %T > MIC required for continuous 1/3‐log, 1/2‐log, and 1‐log reductions were 14.23, 34.45, and 73.44%, respectively, during each 24‐hr treatment period. In conclusion, cefquinome exhibited a potent antibacterial effect against P. multocida. When %T > MIC reached 73.44%, cefquinome exhibited a bactericidal effect against P. multocida after three successive daily administrations.     

Genetic diversity and associated pathology of Pasteurella multocida isolated from porcine pneumonia

Pasteurella multocida is a widespread respiratory pathogen in pigs associated with atrophic rhinitis and contributing to aggravation of the pulmonary lesions. The aims of the present study were to characterize isolates of P. multocida from porcine bronchopneumonia by pulsed-field gel electrophoresis (PFGE), PCR based capsular typing and multilocus sequence typing (MLST) and to compare clonal complexes outlined with the type of histological lung lesions to investigate if a correlation between clonal lineages and lesions might exist. Isolates of P. multocida were obtained from cases of cranioventrally located porcine bronchopneumonia. All lung lesions were described and classified according to histological lesions. A total of 139 isolates, from lung (n=111), pericardial sac (n=21) and kidney (n=7) of 111 pigs were described using PFGE with ApaI as the restriction enzyme. Furthermore, 20 and 29 isolates were characterized by capsular serotyping and multilocus sequence typing, respectively. PFGE demonstrated 15 different clusters showing 50% or more similarity. All selected isolates were of capsular serotype A and only three main sequence types (ST) were detected among the isolates. Associations were not found between histopathology and clonal complexes of P. multocida. In conclusion, PFGE demonstrated a high diversity of genotypes of P. multocida associated with porcine bronchopneumonia. However, isolates obtained mainly belonged to few STs, indicating that isolates of P. multocida associated with porcine bronchopneumonia originates from a limited number of clonal lineages and therefore might have adapted to porcine hosts. No correlation was demonstrated between genotypes and types of lesions, and extra-pulmonary spreading was only rarely demonstrated.     

Actinobacillus pleuropneumoniae culture supernatants interfere with killing of Pasteurella multocida by swine pulmonary alveolar macrophages.

The effect of Actinobacillus pleuropneumoniae culture supernatant on swine pulmonary alveolar macrophage (PAM) functions was studied. The A. pleuropneumoniae culture supernatant was toxic to PAMs when tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH) release assays. Biological activity of the supernatant was ascribed to cytotoxins. Both the LDH and MTT assays were used for measurement of crude A. pleuropneumoniae cytotoxin concentration with good reproducibility. A preparation containing 6,800 toxic units/mL (determined by MTT assay) was used for subsequent experiments. The objective was to study the effect of crude cytotoxin on the ability of swine PAMs to kill Pasteurella multocida. Phagocytosis of opsonized P. multocida type A by PAMs was not efficient. Only 8% of incubated organisms were ingested by noncytotoxin-treated PAMs after 30 min phagocytosis. The bactericidal effect of noncytotoxin-treated PAMs only last for 60 min, after which, the rate of growth of surviving P. multocida exceeded the rate of bacterial killing by PAMs. Complete elimination of P. multocida by PAMs was not observed in this study. A total loss of ability to kill P. multocida by PAMs was seen when the PAMs were pretreated with a high concentration (340 toxic units/mL) of A. pleuropneumoniae cytotoxin. If the PAMs were pretreated with a low concentration (3.4 toxic units/mL) of cytotoxin, a significant reduction in the killing of P. multocida was still observed. The reductions in phagocytosis, phagosome-lysosome fusion (demonstrated using yeast particles of Candida albicans), and oxidative burst (demonstrated by nitro blue tetrazolium reduction (NBT) assay) may have contributed to the impaired killing of P. multocida by PAMs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially.

This study was designed to develop and characterize a swine pneumonic pasteurellosis model by concurrent introduction of Pasteurella multocida type A and Actinobacillus pleuropneumoniae crude cytotoxin. After a series of preliminary experiments, a combination of 4 x 10(9) P. multocida and 4,000 toxic units of A. pleuropneumoniae crude cytotoxin was determined to produce optimal results. A total of 48 pigs were divided into four groups of 12 pigs each. The control group received buffered saline only. Four pigs from each group were randomly selected for necropsy 3, 7 and 14 days postinoculation (PI). Inoculation of pigs with P. multocida and A. pleuropneumoniae cytotoxin (group 1) resulted in moderate to severe pneumonia. Pasteurella multocida was isolated from pneumonic lesions, grossly normal lung, and bronchial lymph nodes of all group 1 pigs throughout the 14 day experimental period. Pathological changes typical of field cases of swine pneumonic pasteurellosis were produced. Pigs inoculated with P. multocida alone (group 2) had pneumonic lesions and P. multocida was reisolated from lungs at three days PI. Pasteurella multocida was not isolated from these pigs at 7 and 14 days PI, except for one pig in which an abscess developed in the thorax. Pulmonary lesions induced by A. pleuropneumoniae crude cytotoxin alone (group 3) were transient and resolved by seven days PI. Group 1 pigs had significantly greater lung lesion volumes than group 2 and 3 pigs at 3, 7 and 14 days PI. Statistical analysis indicated a significant interactive effect of P. multocida and A. pleuropneumoniae cytotoxin on the development of lung lesion volumes at 7 and 14 days PI (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

猪胸膜肺炎放线杆菌的分离和鉴定

某猪场哺乳仔猪及断奶猪临床表现喘气、咳嗽、皮肤发绀以及胸膜肺炎的病理特征，从病猪体内分离出3株菌，根据细菌培养特性、CAMP试验及生化反应等鉴定为猪胸膜肺炎放线杆菌（App)。该菌对小白鼠有一定毒力，回归猪可致死猪只，对常用治疗药有一定的抵抗力，用分离菌制备自家苗可预防本病发生。     

Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds

Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.     

Pharmacokinetic/pharmacodynamic relationship of marbofloxacin against Pasteurella multocida in a tissue‐cage model in yellow cattle

The fluoroquinolone antimicrobial drug marbofloxacin was administered to yellow cattle intravenously and intramuscularly at a dose of 2 mg/kg of body weight in a two‐period crossover study. The pharmacokinetic properties of marbofloxacin in serum, inflamed tissue‐cage fluid (exudate), and noninflamed tissue‐cage fluid (transudate) were studied by using a tissue‐cage model. The in vitro and ex vivo activities of marbofloxacin in serum, exudate, and transudate against a pathogenic strain of Pasteurella multocida (P. multocida) were determined. Integration of in vivo pharmacokinetic data with the in vitro MIC provided mean values for the area under the curve (AUC)/MIC for serum, exudate, and transudate of 155.75, 153.00, and 138.88, respectively, after intravenous dosing and 160.50, 151.00, and 137.63, respectively, after intramuscular dosing. After intramuscular dosing, the maximum concentration/MIC ratios for serum, exudate, and transudate were 21.13, 9.13, and 8.38, respectively. The ex vivo growth inhibition data after intramuscular dosing were fitted to the inhibitory sigmoid E_max equation to provide the values of AUC/MIC required to produce bacteriostasis, bactericidal activity, and elimination of bacteria. The respective values for serum were 17.25, 31.29, and 109.62, and slightly lower values were obtained for transudate and exudate. It is proposed that these findings might be used with MIC₅₀ or MIC₉₀ data to provide a rational approach to the design of dosage schedules which optimize efficacy in respect of bacteriological as well as clinical cures.     

Pharmacokinetic/pharmacodynamic relationship of cefquinome against Pasteurella multocida in a tissue‐cage model in yellow cattle

The cephalosporin antimicrobial drug cefquinome was administered to yellow cattle intravenously (i.v.) and intramuscularly (i.m.) at a dose of 1 mg/kg of body weight in a two‐period crossover study. The pharmacokinetic (PK) properties of cefquinome in serum, inflamed tissue‐cage fluid (exudate), and noninflamed tissue‐cage fluid (transudate) were studied using a tissue‐cage model. The in vitro and ex vivo activities of cefquinome in serum, exudate, and transudate against a pathogenic strain of Pasteurella multocida (P. multocida) were determined. A concentration‐independent antimicrobial activity of cefquinome was confirmed for levels lower than 4 × MIC. Integration of in vivo pharmacokinetic data with the in vitro MIC provided mean values for the time that drug levels remain above the MIC (T > MIC) in serum was 14.10 h after intravenous and 14.46 h after intramuscular dosing, indicating a likely high level of effectiveness in clinical infections caused by P. multocida of MIC 0.04 μg/mL or less. These data may be used as a rational basis for setting dosing schedules, which optimize clinical efficacy and minimize the opportunities for emergence of resistant organisms.     

Minimum inhibitory concentration breakpoints and disk diffusion inhibitory zone interpretive criteria for tilmicosin susceptibility testing against Pasteurella multocida and Actinobacillus pleuropneumoniae associated with porcine respiratory disease.

Tilmicosin is a novel macrolide antibiotic developed for exclusive use in veterinary medicine. Tilmicosin has been approved as a feed premix to control porcine respiratory disease associated with Pasteurella multocida and Actinobacillus pleuropneumoniae. The development of antimicrobial susceptibility testing guidelines for tilmicosin was predicated on the relationship of clinical efficacy studies that demonstrated a favorable therapeutic outcome, on pharmacokinetic data, and on in vitro test data, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The approved breakpoints for the minimum inhibitory concentration dilution testing for both species are resistant, > or = 32 microg/ml, and susceptible, < or = 16 microg/ml. The zone of inhibition interpretive criteria for disk diffusion testing with a 15-microg tilmicosin disk are resistant, < or = 10 mm, and susceptible, > or = 11 mm.     

Long-acting oxytetracycline for control of induced Pasteurella multocida rhinitis in swine

A long-acting oxytetracycline formulation was evaluated for control of rhinitis induced experimentally in pigs with a capsular type A, toxin-negative, low-passage strain of Pasteurella multocida. The pigs were 6 to 7 weeks old and were naturally infected with Haemophilus parasuis. The H parasuis infection was thought to predispose to establishment of P multocida in the nasal cavity. A long-acting oxytetracycline formulation was given IM at the rate of 20 mg/kg, 4 times at 5-day intervals. Medication reduced (P less than 0.05) the severity of turbinate atrophy and the proportion of pigs with P multocida and H parasuis in their nasal cavities. Numbers of colonies of P multocida and H parasuis isolated were also less in medicated pigs.