替米考星对猪传染性胸膜肺炎的治疗试验

为了解替米考星对猪传染性胸膜肺炎的疗效并为临床应用该药提供科学依据,将60头仔猪分为6 组(替米考星高、中、低剂量组及健康、感染和土霉素3 个对照组)进行人工感染试验。结果表明,国产替米考星拌料给药对治疗猪胸膜肺炎放线杆菌病具有明显的效果,可降低死亡率,提高成活率,减少人工感染引起的病理损伤;替米考星中、高剂量组(200 mg/kg饲料、400 mg/kg饲料)疗效尤为显著,因此临床治疗推荐剂量为200 mg/kg饲料,连用7 d。     

Tilmicosin-induced bovine neutrophil apoptosis is cell-specific and downregulates spontaneous LTB4 synthesis without increasing Fas expression

The pathology of bacterial pneumonia, such as seen in the bovine lung infected with Mannheimia haemolytica, is due to pathogen virulence factors and to inflammation initiated by the host. Tilmicosin is a macrolide effective in treating bacterial pneumonia and recent findings suggest that this antibiotic may provide anti-inflammatory benefits by inducing polymorphonuclear neutrophilic leukocyte (PMN) apoptosis. Using an in vitro bovine system, we examined the cell-specificity of tilmicosin, characterized the changes in spontaneous leukotriene B4 (LTB4) synthesis by PMN exposed to the macrolide, and assessed its effects on PMN Fas expression. Previous findings demonstrated that tilmicosin is able to induce PMN apoptosis. These results were confirmed in this study by the Annexin-V staining of externalized phosphatidylserine and the analysis with flow cytometry. The cell-specificity of tilmicosin was assessed by quantification of apoptosis in bovine PMN, mononuclear leukocytes, monocyte-derived macrophages, endothelial cells, epithelial cells, and fibroblasts cultured with the macrolide. The effect of tilmicosin on spontaneous LTB4 production by PMN was evaluated via an enzyme-linked immunosorbent assay. Finally, the mechanisms of tilmicosin-induced PMN apoptosis were examined by assessing the effects of tilmicosin on surface Fas expression on PMN. Tilmicosin-induced apoptosis was found to be at least partially cell-specific, as PMN were the only cell type tested to die via apoptosis in response to incubation with tilmicosin. PMN incubated with tilmicosin under conditions that induce apoptosis spontaneously produced less LTB4, but did not exhibit altered Fas expression. In conclusion, tilmicosin-induced apoptosis is specific to PMN, inhibits spontaneous LTB4 production, and occurs through a pathway independent of Fas upregulation.     

Assessment of the efficacy of tilmicosin phosphate to eliminate Actinobacillus pleuropneumoniae from carrier pigs

The aim of this study was to evaluate the efficacy of in-feed medication with tilmicosin phosphate in order to eliminate or reduce the carriage of Actinobacillus pleuropneumoniae in the tonsils of carrier pigs. Two groups of 6 carrier animals received either a non-medicated feed (control group) or feed medicated with 400 ppm of tilmicosin phosphate (treated group) for 30 d. Three sentinel pigs were then introduced in each group and left for 29 d. The presence of A. pleuropneumoniae in tonsils was monitored using several techniques, including polymerase chain reaction (PCR). At the end of the treatment all of the control animals, but only 1 treated pig, were positive by PCR from tonsillar surface material. However, at necropsy, all control and most treated animals, as well as 1 sentinel animal, in both groups were positive by PCR from whole tonsils. In conclusion, under the experimental conditions, in-feed treatment with 400 ppm of tilmicosin phosphate significantly reduced the presence of A. pleuropneumoniae on the surface of tonsils but was unable to completely eliminate the organism from deeper tonsillar tissues and to prevent bacterial shedding by carrier animals.     

Early in vivo interactions of Actinobacillus pleuropneumoniae with tonsils of pigs.

Twenty gnotobiotic piglets were inoculated with 5 x 10(8) colony forming units of an Actinobacillus pleuropneumoniae biotype 1-serotype 9 strain onto their tonsils. Five other piglets (controls) were inoculated with phosphate-buffered saline solution. Pigs were euthanized at 30 min, 90 min, 180 min, 6 h, 9 h, 12 h or 24 h after inoculation. At necropsy, samples were taken from the tonsils for bacteriological, histological, immuno-histochemical and electron microscopical examination. A. pleuropneumoniae was isolated from tonsils of all the infected pigs, but not from tonsils of the control pigs. Early after inoculation bacteria were mainly associated with the stratified squamous epithelium and detached epithelial cells. Vacuolization and desquamation of the epithelium was observed and many transmigrating neutrophils were present. At later times after inoculation, bacteria were found closely associated with the crypt-walls and with detached cells present in the crypts. A strong neutrophil migration was observed mainly in the deeper parts of the crypts. It is concluded that attachment of A. pleuropneumoniae to tonsillar epithelial cells probably constitutes a first step in establishing bacteria at this body site.     

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.     

Effect of tilmicosin on chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages

OBJECTIVE: To evaluate chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages (AM) exposed to tilmicosin. ANIMALS: 12 healthy calves and 12 healthy pigs. PROCEDURES: Lungs were obtained immediately after euthanasia; AM were collected by means of bronchoalveolar lavage and density gradient centrifugation. Chemotactic activity was evaluated by exposing AM to lipopolysaccharide or macrophage inhibitory peptide during incubation with tilmicosin. Phagocytic activity was evaluated by incubating AM with tilmicosin for 24 hours and then with tilmicosin-resistant Salmonella serotype Typhimurium. Bactericidal activity was evaluated by incubating AM with tilmicosin (0, 10, or 20 microg/ml for bovine AM; 0 or 10 microg/ml or 10 microg/ml but washed free of tilmicosin for porcine AM) and then with Mannheimia haemolytica (bovine AM) or with Actinobacillus pleuropneumoniae or Pasteurella multocida (porcine AM). RESULTS: Tilmicosin had no significant effects on chemotactic or phagocytic activities of bovine or porcine AM. The time-course of bactericidal activity was best described by polynomial equations. Time to cessation of bacterial growth and area under the time versus bacterial number curve were significantly affected by incubation of AM with tilmicosin. CONCLUSIONS AND CLINICAL RELEVANCE: Results show that bactericidal activity of bovine and porcine AM was enhanced by tilmicosin, but not in proportion to the reported ability of AM to concentrate tilmicosin intracellularly. With or without exposure to tilmicosin, the time-course of bactericidal activity of bovine AM against M haemolytica and of porcine AM against A pleuropneumoniae or P multocida was too complex to be reduced to a simple linear equation.     

Role of haemophilus pleuropneumoniae lipopolysaccharide endotoxin in the pathogenesis of porcine Haemophilus pleuropneumonia

Intact Haemophilus pleuropneumoniae cells (strain Shope 1, serotype 1), highly purified lipopolysaccharide (LPS) obtained from this strain of H pleuropneumoniae, as well as from Escherichia coli O111:B4, filter-sterilized H pleuropneumoniae cell-free culture supernatant fluid, and heat-inactivated supernatant fluid were given intranasally to CF1 mice and intratracheally to pigs. Pulmonary lesions induced by H pleuropneumoniae in mice were similar to those induced by H pleuropneumoniae in pigs. Histologically, lungs of mice and pigs killed 1 or 2 days after inoculation with 200 micrograms of highly purified H pleuropneumoniae LPS had lesions similar to one another and were similar to those in mice and pigs given intact H pleuropneumoniae, except that little or no necrosis or hemorrhage was observed. In mice killed 1 or 2 days after inoculation of 200 micrograms of E coli O111:B4 LPS, pulmonary lesions were similar to those in mice given H pleuropneumoniae LPS. Pulmonary lesions in mice given cell-free culture supernatant fluid obtained from a midlog-phase growth culture of H pleuropneumoniae cultivated in a chemically defined medium were severe and consisted of neutrophil infiltration and extensive necrosis. In mice, the heat-inactivated supernatant fluid produced mild lesions that consisted of foci of neutrophil aggregation and no necrosis. Extensive necrosis observed in lesions caused by cell-free culture supernatant fluid could be attributed to the action of a heat-labile component, perhaps by the extracellular heat-labile hemolysin produced by H pleuropneumoniae cultivated in chemically defined medium. A LPS endotoxin and a heat-labile factor may be involved in the pulmonary lesion development in the acute phase of porcine Haemophilus pleuropneumonia.     

Tilmicosin reduces lipopolysaccharide-stimulated bovine alveolar macrophage prostaglandin E(2) production via a mechanism involving phospholipases

Tilmicosin is a potent antimicrobial with broad-spectrum activity against the bacterial agents involved in the bovine respiratory disease complex. Recent studies indicate that in addition to being bactericidal, tilmicosin is capable of modulating inflammation in the lung. A series of experiments were designed to determine whether tilmicosin alters alveolar macrophage-prostaglandin E(2) (PGE(2)) production induced by Escherichia coli (O55:B5) lipopolysaccharide (LPS). Twenty-two healthy Holstein bull calves were used to study the effects of LPS-induced PGE(2) production of alveolar macrophages after in vivo or in vitro treatment with tilmicosin. In Experiment 1, tilmicosin was given by subcutaneous injection (15 mg/kg) twice, 48 hours apart, to four calves; four control calves received no treatment. Twenty-four hours after the second treatment, alveolar macrophages were stimulated with LPS in vitro. In Experiment 2, alveolar macrophages from five untreated calves were harvested and treated in vitro with tilmicosin, followed by LPS stimulation. In Experiment 3, the ability of in vitro tilmicosin treatment to alter the expression of LPS-induced cyclooxygenase-2 (COX-2) mRNA was evaluated. In Experiments 4 and 5, secretory phospholipase A(2) activity was examined in untreated calves. Treatment of calves with tilmicosin resulted in reduced LPS-induced alveolar macrophage PGE(2) production. Similar reductions in PGE(2) by LPS-stimulated alveolar macrophages after in vitro tilmicosin treatment were noted. This in vitro tilmicosin treatment was not associated with reduction of the expression of LPS-induced COX-2. Alveolar macrophage phospholipase A(2) activity induced by LPS was significantly reduced by prior tilmicosin treatment in vitro. Tilmicosin (in vivo and in vitro) appears to reduce the PGE(2) eicosanoid response of LPS-stimulated alveolar macrophages by reducing the in vitro substrate availability without altering in vitro COX-2 mRNA expression.     

Comparative virulence of NAD-dependent and NAD-independent Actinobacillus pleuropneumoniae strains.

The virulence of a NAD-independent Actinobacillus pleuropneumoniae serotype 2 strain and NAD-dependent serotype 2, 3 and 9 strains was compared under experimental conditions. Hysterectomy-derived piglets were inoculated endobronchially with 50-500 cfu of these strains. All 23 piglets inoculated with the NAD-dependent strains developed acute disease within 12 hours post inoculation. Twenty-two of these piglets died within 24 hours after the first clinical signs. Three of nine piglets inoculated with the NAD-independent strain did not develop clinical disease. In the other six piglets, disease signs were similar as in the piglets inoculated with the NAD-dependent strains. No differences in clinical disease were observed between colostrum deprived piglets and piglets that obtained colostrum from a SPF sow.     

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

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

Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs.

In order to demonstrate the possible role of aerosol in the transmission of Actinobacillus pleuropneumoniae, an experiment including 18 specific pathogen-free (SPF), 10-week-old piglets, randomly distributed into 2 adjacent units, was carried out. In these facilities, air was forced through absolute filters to prevent any contact with infectious agents. During the first 6 d post inoculation, the 2 units were connected by a rectangular opening and the air circulation was forced by the ventilation system from unit A (inoculated pigs) to unit B (non-inoculated pigs). The A. pleuropneumoniae strain (biovar 1 serovar 9) was isolated in France from an outbreak of porcine pleuropneumonia. Two different infecting doses, 10(7) cfu/animal and 10(8) cfu/animal, were inoculated by intranasal route in 6 pigs of unit A. The infection spread quickly from the inoculated pigs to the non-inoculated pigs. Clinical signs were acute during the 4 d post inoculation: hyperthermia, respiratory distress and, sometimes, death (6 pigs of the unit A and 2 pigs of the unit B). All pigs seroconverted against A. pleuropneumoniae serovar 9 within 2 weeks. Lung lesions were severe: fibrinous pleurisy and lung hemorrhages in the acute stage, pleural adherences and focal pulmonary necrosis in the chronic stage. Actinobacillus pleuropneumoniae was isolated from the tonsils and/or lungs in 16 animals. It could be also isolated from the air of the experimental unit. This study showed that A. pleuropneumoniae was readily transmitted through aerosol over a distance of at least 2.5 m.     

The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets

Colonisation of type D Pasteurella multocida was studied in five groups of seven SPF piglets each. Piglets of Group 1 were kept together with seven 5-week-old piglets obtained from a large herd infected with toxigenic P. multocida for 16 weeks (contact infection). These piglets were made free from toxigenic Bordetella bronchiseptica by local immunisation. Piglets of Group 2 were inoculated with 5 x 10(7) colony-forming units (cfu) of P. multocida washed from the nasal mucosa of piglets free from toxigenic B. bronchiseptica with fetal calf serum. Piglets of Group 3 were inoculated intranasally with 5 x 10(7) cfu of P. multocida washed from yeast-extract proteose-peptone cystine (YPC)-blood agar with fetal calf serum. Piglets of Group 4 were inoculated with 5 x 10(7) cfu of P. multocida grown in a YPC-based broth without blood. Piglets of Group 5 served as controls. The piglets of Group 1 did not contract P. multocida infection from infected contact piglets. After a single inoculation one of four, while after three inoculations two of three piglets of Group 2 became infected by P. multocida. After a single inoculation none of four, while after three inoculations one of three piglets of Group 3 were colonised by P. multocida. Both single and repeated inoculation failed in piglets of Group 4.     

Pulmonary disposition of tilmicosin in foals and in vitro activity against Rhodococcus equi and other common equine bacterial pathogens

The objectives of this study were to determine the serum and pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against Rhodococcus equi and other common bacterial pathogens of horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of body weight) was administered to seven healthy 5- to 8-week-old foals by the intramuscular route. Concentrations of tilmicosin were measured in serum, lung tissue, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils. Mean peak tilmicosin concentrations were significantly different between sampling sites with highest concentrations measured in blood neutrophils (66.01+/-15.97 microg/mL) followed by BAL cells (20.1+/-5.1 microg/mL), PELF (2.91+/-1.15 microg/mL), lung tissue (1.90+/-0.65 microg/mL), and serum (0.19+/-0.09 microg/mL). Harmonic mean terminal half-life in lung tissue (193.3 h) was significantly longer than that of PELF (73.3 h), bronchoalveolar cells (62.2 h), neutrophils (47.9 h), and serum (18.4 h). The MIC90 of 56 R. equi isolates was 32 microg/mL. Tilmicosin was active in vitro against most streptococci, Staphylococcus spp., Actinobacillus spp., and Pasteurella spp. The drug was not active against Enterococcus spp., Pseudomonas spp., and Enterobacteriaceae.     

Pharmacokinetics of tilmicosin after oral administration in swine

OBJECTIVE: To determine the pharmacokinetics of tilmicosin after oral administration of a single dose of tilmicosin base in swine. ANIMALS: 10 healthy swine. PROCEDURE: Tilmicosin base was administered via stomach tube at a single dose of 20 mg/kg (n = 5) or 40 mg/kg (5). Blood samples were obtained from a jugular vein immediately before and at 10, 20, and 30 minutes and 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 hours after administration of tilmicosin. Tilmicosin concentrations in serum were quantified by use of a high-performance liquid chromatography procedure with UV light. Data for tilmicosin concentrations versus time were analyzed by use of compartmental and noncompartmental methods. RESULTS: Tilmicosin concentrations in serum decreased in a biexponential manner after oral administration. Mean +/- SD values for absorption half-lives were 1.49 +/- 0.23 hours and 1.64 +/- 0.40 hours, distribution half-lives were 2.96 +/- 0.58 hours and 3.20 +/- 0.76 hours, elimination half-lives were 25.26 +/- 8.25 and 20.69 +/- 5.07 hours, peak concentrations were 1.19 +/- 0.30 microg/mL and 2.03 +/- 0.28 microg/mL, and time to peak concentrations was 3.12 +/- 0.50 hours and 3.48 +/- 0.77 hours after oral administration of tilmicosin base at a single dose of 20 or 40 mg/kg, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In swine, tilmicosin was rapidly absorbed and slowly eliminated after oral administration of a single dose of tilmicosin base powder.     

Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1

Sixteen 3-week-old calves were intratracheally inoculated with Mycoplasma bovis. Follow-up consisted of regular bronchoalveolar lavages (BALs) and clinical examinations. Animals were slaughtered from 4 to 21 days after inoculation. Counts were made of the mycoplasmas and other bacteria systematically isolated from the BAL liquids and lung lobes after slaughter. On the 6th day, spectinomycin 20mg/kg was given intramuscularly in three repeated doses at 24h intervals to six randomly chosen calves. All animals had developed a persistent M. bovis infection with a maximum BAL count on the 6th day (start of treatment). Co-occuring Pasteurella multocida infection was found in most animals with a maximum rate on the 14th day. The extent of lung surface lesions varied widely (0-64%) but was greater in the later slaughtered calves. Average counts of M. bovis and P. multocida in the BAL liquids were lower in treated calves than in untreated ones but the difference was not statistically significant. However, M. bovis and P. multocida counts in the lungs of the treated group were significantly lower than in the untreated group (p=0.003 and 0.009, respectively).     

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.     

Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28DPI compared to the other groups. At 10 and 15DPI, animals in the highly virulent group had significantly higher concentrations of TNF-alpha in BAL fluid. IL-1beta concentration in this group was higher at 5 and 28DPI compared to the other groups. From 10DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.     

Intracellular accumulation,subcellular distribution and efflux of tilmicosin in swine phagocytes

Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease. As the concentrations of tilmicosin are generally low in swine lung tissue, the interaction of tilmicosin with three types of swine phagocytes (monocyte-macrophages, alveolar macrophages, and neutrophils) was evaluated to provide an understanding of clinical efficacy. After incubation with radiolabelled tilmicosin, uptake was determined and expressed as the ratio of the intracellular (Ci) to the extracellular (Ce) drug concentration (Ci/Ce). Tilmicosin was avidly accumulated by the swine phagocytes (Ci/Ce 48–69 at 4 h incubation) with 51 to 85% localized in the lysosomes. Uptake was dependent on cell viability, temperature and pH, but was not influenced by the metabolic inhibitors, sodium cyanide or potassium fluoride. However, lipopolysaccharide (LPS) exposure increased tilmicosin uptake by the swine phagocytes. In neutrophils, upon removal of extracellular tilmicosin, 60% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in drug-free medium, 25% remained cell-associated. In contrast, after 4 h of incubation in drug-free medium, 60% and 45% of tilmicosin remained cell-associated, within alveolar macrophages and monocyte-derived macrophages, respectively. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme and β-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro interactions of tilmicosin with swine phagocytes suggest an integral role in effecting clinical efficacy.     

Pharmacokinetics of tilmicosin in equine tissues and plasma

The macrolide antibiotic tilmicosin has potential for treating bacterial respiratory tract infections in horses. A pharmacokinetic study evaluated the disposition of tilmicosin in the horse after oral (4 mg/kg) or subcutaneous (s.c.) (10 mg/kg) administration. Tilmicosin was not detected in equine plasma or tissues after oral administration at this dose. With s.c. injection, tilmicosin concentrations reached a maximum concentration of approximately 200 ng/mL in the plasma of the horses. Tilmicosin concentrations in plasma persisted with a mean residence time (MRT) of 19 h. Maximum tissue residue concentrations (C(max)) of tilmicosin measured in equine lung, kidney, liver and muscle tissues after s.c. administration were 2784, 4877, 1398, and 881 ng/g, respectively. The MRT of tilmicosin in these tissues was approximately 27 h. Subcutaneous administration of tilmicosin resulted in severe reactions at the injection sites.     

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.