Effect of apo-lactoferrin on leukotoxin and outer membrane vesicles of Mannheimia haemolytica A2.

Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2-8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.

Antimicrobial resistance and genetic characterization of fluoroquinolone-resistant Mannheimia haemolytica isolates from cattle with bovine pneumonia.

Antimicrobial susceptibility and molecular characterization of quinolone-resistant Mannheimia haemolytica was conducted. The antimicrobial susceptibility of 229 M. haemolytica isolates which were obtained from cattle with bovine respiratory disease during the period 1984-2006, was determined using 14 antimicrobial agents. Of the 229 isolates, 114 (49.8%) were resistant to at least one agent and resistance rates ranged from 4.8% to 31.4%. Resistance rates for dihydrostreptomycin, oxytetracycline, doxycycline, ampicillin, amoxicillin, thiamphenicol, kanamycin chloramphenicol, nalidixic acid, enrofloxacin, and danofloxacin were 31.4%, 20.5%, 18.3%, 19.2%, 16.6%, 10.9%, 11.4%, 10.5%, 17.0%, 4.8% and 4.8%, respectively. The nucleotide sequences of the quinolone resistance-determining regions of the gyrA and parC genes of nalidixic acid-resistant M. haemolytica were determined. All nalidixic acid-resistant strains possessed at least one amino acid substitution in each of the GyrA and ParC fragments investigated. These results suggest that M. haemolytica require at least one amino acid substitution in both GyrA and ParC in order to attain significant levels of resistance to quinolones. All fluoroquinolone-resistant isolates belonged to serotype 6, and their genotype by PFGE analysis was identical. This result indicates that fluoroquinolone-resistant M. haemolytica strains have clonally expanded.

Tulathromycin: an overview of a new triamilide antibiotic for livestock respiratory disease.

Tulathromycin is a novel triamilide antimicrobial that has been approved for use in the treatment and prevention of bovine respiratory disease and the treatment of swine respiratory disease in the European Union and the United States. The agent penetrates gram-negative bacteria well, and it exhibits mixed bacteriostatic and bactericidal activity. Tulathromycin is formulated as a ready-to-use, sterile aqueous solution, and the packaged concentration of 100 mg tulathromycin/ml allows low-volume dosing. This agent is characterized by rapid absorption from the injection site, extensive distribution to tissue, and slow elimination, thereby providing high, prolonged drug concentration in the lungs. Studies show that a single dose of tulathromycin is effective in treating cattle and swine with respiratory disease and in preventing high-risk cattle from developing respiratory disease.

Efficacy of tulathromycin injectable solution for the treatment of naturally occurring Swine respiratory disease.

Tulathromycin, a novel triamilide antimicrobial, was evaluated for treatment of swine respiratory disease (SRD) in field efficacy studies involving 720 pigs in six North American swine herds. In each study, feeder pigs with clinical SRD were randomly assigned in equal numbers to a group treated with tulathromycin given as a single injection at 2.5 mg/kg of body weight or to a saline-treated control group. Four of the studies included a third group treated with ceftiofur sodium for 3 consecutive days at 3 mg/kg of body weight. Pigs were treated on day 0 and evaluated for treatment response on day 7. In each study, 10 or more nontreated pigs and saline-treated pigs that did not respond to treatment underwent necropsies to obtain lung samples that were evaluated for SRD pathogens. The overall cure rate was 46.4% for saline-treated pigs, 71.1% for tulathromycin-treated pigs, and 63.1% for ceftiofur-treated pigs. The cure rate for tulathromycin-treated pigs was significantly higher than for saline-treated pigs (P = .0116). Mortality from SRD occurred in 24 control pigs, seven tulathromycin-treated pigs, and one ceftiofur-treated pig. The mortality rate was significantly lower for both the tulathromycin- and ceftiofur-treated pigs compared with those treated with saline (P = .0148 and P = .0195, respectively). Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis, and Mycoplasma hyopneumoniae, bacteria commonly associated with SRD, were isolated from SRD-affected pigs. Under field conditions, tulathromycin injectable solution given as a single IM dose of 2.5 mg/kg of body weight was safe and effective in the treatment of SRD.

Dose determination and confirmation of a long-acting formulation of ceftiofur (ceftiofur crystalline free acid) administered subcutaneously for the treatment of bovine respiratory disease.

The objective of this work was to determine and confirm an effective dose of ceftiofur crystalline free acid sterile oil suspension (CCFA-SS, 100 mg ceftiofur equivalents (CE)/mL], a long-acting single-administration ceftiofur formulation, for the treatment of the bacterial component of bovine respiratory disease (BRD). Study 1 was a dose determination study that used an intratracheal Mannheimia haemolytica (Pasteurella haemolytica) challenge model to evaluate single-administration doses of CCFA-SS at 0.0, 1.1, 2.2, 3.3, 4.4 or 5.5 mg CE/kg body weight (BW) for the treatment of BRD. Data from this study were used to select doses for field testing in three multi-location clinical studies. In Study 2, the efficacy of a single administration dose of CCFA-SS at 4.4 mg CE/kg BW was compared with a negative control for the treatment of naturally occurring BRD in feedlot cattle. Treatments were administered when uniform clinical signs of BRD were present. Study 3 used a design similar to Study 2, and compared single-administration doses of CCFA-SS at 3.0 or 4.4 mg CE/kg BW with the positive-control tilmicosin (Micotil(R) 300 Injection, Elanco Animal Health) at 10 mg/kg BW. Study 4 compared the efficacy of single doses of CCFA-SS of 1.1-8.8 mg CE/kg BW with tilmicosin at 10 mg/kg BW. A total of 1176 cattle were included in these clinical studies. In Study 1, a dose of 4.55 mg CE/kg BW was determined to be effective. This was rounded to 4.4 mg CE/kg for field testing. In Study 2, a single dose of CCFA-SS at 4.4 mg CE/kg BW had a higher treatment success rate on day 14 (61%) than negative controls (26%, P < 0.01). However, in Study 3 this dose was judged to be at the beginning of an efficacious dose range for the treatment of BRD when compared with tilmicosin. In Study 4, day 28 treatment success rates were higher for CCFA-SS at 4.4-8.8 CE/kg BW than for tilmicosin (P=0.002) or the noneffective CCFA-SS dose of 1.1 mg CE/kg BW (P < 0.001). Based on decision criteria for Study 4, the effective dose was determined to be 4.4-5.5 mg CE/kg BW. These clinical studies demonstrated that a single dose of CCFA-SS (100 mg CE/mL) administered subcutaneously (s.c.) in the neck at 4.4-5.5 mg CE/kg BW is an effective treatment for BRD in feedlot cattle. However, this route of administration is no longer being considered for this formulation because of the ceftiofur residues that are present at the injection site for extended periods of time.

The clinical efficacy of enrofloxacin in the treatment of experimental bovine pneumonic pasteurellosis.

The clinical efficacy of enrofloxacin was tested in calves with experimentally induced pneumonic pasteurellosis. A strain of Pasteurella haemolytica, biotype A, serotype 1 (P. haemolytica A1), which had been isolated from an outbreak of pneumonic pasteurellosis in feedlot calves, was used to induce the disease in 24 eight-month-old calves. Each animal received, by intratracheal injection, 6 x 10(11) colony forming units of P. haemolytica A1 in a four-hour log phase culture. Twelve similar animals were kept as non-infected controls (Negative Control group). Treatment of the infected animals commenced 40 h after infection and was as follows: 12 animals each received 2.5 mg/kg enrofloxacin subcutaneously and 12 animals each received 5 ml sterile saline intramuscularly (Positive Control group). All treatments were given once daily for three consecutive days. Clinical examinations were performed on all animals once daily, starting prior to infection and continuing until 12 d post-infection. The parameters evaluated were rectal temperature, habitus (attitude), ocular mucous membrane congestion and abnormal sounds on lung auscultation. On day 14 post-infection, all animals were killed and their lung lesions (if any) estimated as the percentage involvement of each pair of lungs. The only statistically significant (P > or = 0.05) differences observed were between the Negative Control group and the Positive Control group. Noticeable differences were seen between the enrofloxacin-treated group and the Positive Control group, but they were not statistically significant (P > 0.05). The average lung lesion score (pneumonic lesions as a percentage of total lung volume) for the Positive Control group was 12.1% and that of the enrofloxacin-treated group, 8.4%. This difference was not statistically significant (P > 0.05).

Economic, clinical and functional consequences of a treatment using metrenperone during an outbreak of shipping fever in cattle.

Eighteen of 91 seven- to nine-month-old Belgian white and blue double-muscled male fattening cattle developed typical signs of shipping fever. They were all injected intramuscularly once a day for three days with 5 mg/kg of enrofloxacin, and in addition nine selected at random were injected intramuscularly five times at 12 hour intervals with 0.1 mg/kg of metrenperone, a 5-hydroxytryptamine blocker, the other nine receiving a placebo. During the outbreak of shipping fever metrenperone showed effective antipyretic properties, and all the calves treated with it made a complete recovery. Moreover, during the 360 day fattening period following the outbreak, the cattle treated with metrenperone gained on average 45.4 kg more weight than the control cattle.

Use of an indwelling bronchial catheter model of bovine pneumonic pasteurellosis for evaluation of therapeutic efficacy of various compounds.

A model of bovine pneumonic pasteurellosis, using an indwelling bronchial catheter for inoculation and subsequent lavage of a single main stem bronchus of the lung, was evaluated in a preliminary efficacy trial of an experimental therapeutic compound. Inoculation of 10(7) Pasteurella haemolytica organisms into the bronchus consistently induced a focal pneumonic lesion with typical morphology of pneumonic pasteurellosis in the left or right caudal lung lobe. The experimental treatment caused significant (P less than 0.05) reduction in lung lesion volume, compared with that of a saline-treated control. It also caused significant (P less than 0.05) reduction in lavage fluid bacterial counts at 48 hours after inoculation, compared with counts in the controls. The inflammatory cell count and the percentage of neutrophils increased markedly in lavage fluids 8 hours after inoculation, but differences were not detected between treatments. Significant differences between treatments were not found in clinical signs, rectal temperature, or histologic changes. This model appears to be a sensitive indicator of treatment efficacy and has the advantage over previous models of pneumonic pasteurellosis of allowing sequential monitoring of the primary lesion site.

In vitro assessment of the efficacy of erythromycin in combination with oxytetracycline or spectinomycin against Pasteurella haemolytica.

The effects of combining erythromycin (Ery) with oxytetracycline (Oxy) or spectinomycin (Sp) on Pasteurella haemolytica were evaluated in vitro using the chessboard (checkerboard) technique. These combinations were selected because all are drugs widely used in bovine respiratory disease treatment, and they represent possible sequential or complementary mechanisms of action. Using the recommended breakpoints of greater than 4 micrograms/ml for Ery, 16 micrograms/ml for Oxy, and 32 micrograms/ml for Sp, of the 33 P. haemolytica isolates, 32 were resistant to Oxy, 27 to Sp, and 14 to Ery. Based on the fractional inhibitory concentration index, Ery and Oxy in combination were synergistic or additive against 32 of 33 isolates. The combination of Ery and Sp was synergistic or additive against 27 of 33 isolates. No instances of antagonism were seen. When the effects were considered within the context of therapeutically achievable serum/tissue concentrations, the effects of Ery and Oxy in combination were only marginal. Thus, against P. haemolytica isolates, Ery and Sp appeared to represent an effective antimicrobial combination, whereas Ery and Oxy were only of marginal efficacy as a combination.

Effect of enrofloxacin therapy on shipping fever pneumonia in feedlot cattle.

The effect of enrofloxacin therapy was investigated in 110 male double-muscled cattle weighing 275 +/- 3 kg, during a spontaneous outbreak of shipping fever occurring 11 +/- 2 days after they arrived in the feedlot. Forty-six diseased animals were divided randomly into three groups A, B and C, containing 17, 19 and 10 animals, respectively; the animals in group A were injected intramuscularly once daily for three consecutive days with 2.5 mg/kg of enrofloxacin, those in group B with 5 mg/kg of enrofloxacin and those in group C with 10 mg/kg of oxytetracycline. Clinical, serological, production and respiratory functional observations were recorded. The animals were clinically cured after the three day treatment except for three in group A and two in group C. These five animals made a clinical recovery after a three day booster treatment with a dose of 5 mg/kg enrofloxacin. The changes in respiratory gas exchange values induced by shipping fever were completely reversed 15 days later, suggesting that there had been no irreversible lung damage. The daily weight gains and the arterial blood gas values of the three groups of treated cattle were not significantly different. The high efficacy of the low dosage of enrofloxacin in this clinical syndrome may be explained by its antibacterial activity against Pasteurella species and Mycoplasma species. This field trial supports the in vitro studies which suggested than enrofloxacin is an appropriate therapy in cases of shipping fever.