A microbiological assay to estimate the antimicrobial activity of parenteral tildipirosin against foodborne pathogens and commensals in the colon of beef cattle and pigs.

Tildipirosin (TIP) is a novel 16-membered-ring macrolide authorized for the treatment of bovine and swine respiratory disease. The pH dependency of macrolide antimicrobial activity is well known. Considering that the pH in the colon contents of growing beef cattle and pigs is usually below pH 7.0, the minimum inhibitory concentrations (MIC) of TIP against foodborne bacterial pathogens such as Campylobacter (C.) coli, C. jejuni and Salmonella enterica and commensal species including Enterococcus (E.) faecalis, E. faecium and Escherichia coli were determined under standard (pH 7.3 ± 1) or neutral as well as slightly acidic conditions. A decrease in pH from 7.3 to 6.7 resulted in an increase in MICs of TIP. Except for the MICs > 256 µg/mL observed in the resistant subpopulation of the C. coli and the Enterococcus species, the MIC ranges increased from 2-8 µg/mL to 64-> 256 µg/mL for Salmonella enterica and E. coli, from 8-16 µg/mL to 32-128 µg/mL for the two Campylobacter species, and from 4-32 µg/mL to 128-> 256 µg/mL for both Enterococcus species. To estimate the antimicrobial activity of TIP in the colon contents of livestock during recommended usage of the parenterally administered TIP (Zuprevo(®) ), and to compare this with the increased MICs at the slightly acidic colonic pH, we developed and validated a microbiological assay for TIP and used this to test incurred faecal samples collected from cattle and pigs. Microbiological activity of luminal TIP was determined in aqueous supernatants from diluted faeces, using standard curves produced from TIP-spiked faecal supernatants. The limit of quantification (LOQ) for TIP was 1 µg/mL (ppm). In a cattle study (n = 14), 3 of 28 faecal samples collected 24 and 48 h post-treatment were found to contain TIP above the LOQ (concentrations of 1.3-1.8 ppm). In another cattle study (n = 12) with faecal samples collected at 8, 24 and 48 h post-treatment, TIP concentrations were above the LOQ in 4 of the 8 h samples (1.2-2.6 ppm) and one of the 24-h samples (1.3 ppm). In a pig study (n = 12) with faecal samples collected 24, 48 and 72 h post-treatment, only one sample contained TIP above the LOQ (concentration 1.5 ppm). In another pig study (n = 12), with samples collected at 8, 24 48 and 96 h post-treatment, TIP concentrations were above the LOQ in one 8-h sample (1.1 ppm) and two 24-h samples (2.3 and 2.5 ppm). None of the 48-h and 96-h samples from these 4 studies contained measurable TIP concentrations. Thus, in cattle and pigs, only a small fraction of faecal samples collected up to 24 h postdosing contained measurable microbiologically active TIP, with its maximum limited to 2.6 µg/mL. This is several log2 dilution steps below the MICs of TIP against foodborne pathogens and commensals collected under acidic conditions comparable with those in the colonic contents and may explain a lack of intestinal dysbacteriosis with parenteral tildipirosin in livestock.

Pharmacokinetics of tildipirosin in porcine plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae.

The pharmacokinetics of tildipirosin (Zuprevo(®) 40 mg/mL solution for injection for pigs), a novel 16-membered-ring macrolide for the treatment for swine respiratory disease (SRD), was investigated in studies collecting blood plasma and postmortem samples of lung tissue and bronchial fluid (BF) from swine. In view of factors influencing the in vitro activity of macrolides, and for the interpretation of tildipirosin pharmacokinetics in relation to minimum inhibitory concentrations (MIC), additional experiments were conducted to study the effects of pH, carbon dioxide-enriched atmosphere, buffers, and serum on tildipirosin MICs for various reference strains and Actinobacillus (A.) pleuropneumoniae field isolates. After single intramuscular (i.m.) injection at 4 mg/kg body weight, maximum plasma concentration (Cmax) was 0.9 µg/mL observed within 23 min (Tmax ). Mean residence time from the time of dosing to the time of last measurable concentration (MRTlast) and terminal half-life (T1/2) both were about 4 days. A dose-response relationship with no significant sex effect is observed for area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUClast) over the range of doses up to 6 mg/kg. However, linear dose proportionality could not be proven with statistical methods. The time-concentration profile of tildipirosin in BF and lung far exceeded that in blood plasma. In lung, tildipirosin concentrations reached 3.1 µg/g at 2 h, peaked at 4.3 µg/g at day 1, and slowly declined to 0.8 µg/g at day 17. In BF, tildipirosin levels were 14.3, 7.0, and 6.5 µg/g at days 5, 10, and 14. T1/2 in lung was ∼7 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination. Culture media pH and carbon dioxide-enriched atmosphere (CO2 -EA) had a marked impact on in vitro activity of tildipirosin in reference strains of various rapidly growing aerobic and fastidious bacteria including Histophilus (H.) somni ATCC 700025 and A. pleuropneumoniae ATCC 27090. For A. pleuropneumoniae ATCC 27090 testing conditions without CO2 -EA resulted in reduced acidification of culture media pH and a reduction in the minimum inhibitory concentrations compared to standard in vitro test conditions by 2 log2 dilution steps (4-fold) from 8 to 2 µg/mL. Supplementary buffering of standard culture media resulted in a reduction in the A. pleuropneumoniae (n = 8) MIC range by 4 log2 dilution steps (16-fold) from 8-16 to 0.5-1 µg/mL. Incremental supplementation of culture media with 50% serum resulted in noticeable shifts to lower minimum or maximum MICs by at least 2 log2 dilution steps (≥4-fold) in all aerobic and fastidious reference strains tested except for Pasteurella (P.) multocida. The MIC of A. pleuropneumoniae ATCC 27090 decreased by 2-4 log2 dilution steps (4 to 16-fold) from 8 to 0.5-2 µg/mL when 50% serum was added to the standard assay. Considering a higher presence of serum and the rather neutral pH conditions maintained in vivo, it is suggested to take the influence of these factors on in vitro activity into account when interpreting tildipirosin MICs for A. pleuropneumoniae in relation to pharmacokinetics.

Pharmacokinetics of tildipirosin in bovine plasma, lung tissue, and bronchial fluid (from live, nonanesthetized cattle).

The pharmacokinetics of tildipirosin (Zuprevo(®) 180 mg/mL solution for injection for cattle), a novel 16-membered macrolide for treatment, control, and prevention of bovine respiratory disease, were investigated in studies collecting blood plasma, lung tissue, and in vivo samples of bronchial fluid (BF) from cattle. After single subcutaneous (s.c.) injection at 4 mg/kg body weight, maximum plasma concentration (C(max)) was 0.7 µg/mL. T(max) was 23 min. Mean residence time from the time of dosing to the time of last measurable concentration (MRT(last)) and terminal half-life (T(1/2) ) was 6 and 9 days, respectively. A strong dose-response relationship with no significant sex effect was shown for both C(max) and area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUC(last) ) over the range of doses up to 6 mg/kg. Absolute bioavailability was 78.9%. The volume of distribution based on the terminal phase (V(z)) was 49.4 L/kg, and the plasma clearance was 144 mL/h/kg. The time-concentration profile of tildipirosin in BF and lung far exceeded those in blood plasma. In lung, tildipirosin concentrations reached 9.2 µg/g at 4 h, peaked at 14.8 µg/g at day 1, and slowly declined to 2.0 µg/g at day 28. In BF, the concentration of tildipirosin reached 1.5 and 3.0 µg/g at 4 and 10 h, maintained a plateau of about 3.5 µg/g between day 1 and 3, and slowly declined to 1.0 at day 21. T(1/2) in lung and BF was approximately 10 and 11 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination.

Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study.

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98-101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

[Inhibition of bone tissue formation by propane-2,2-diphosphonate]. / Hemmung de Knochengewebsbildung durch Propan-2,2-diphosphonat.

Among other diphosphonates here described propane-2,2-diphosphonate displays specific effects on bone-turnover. In contrast to other diphosphonates propane-2,2-diphosphonate inhibits especially different enzymes of growing bone and therefore inhibits formation of bone. The indication for the clinical use follows from that effect: pathologic states with increased bone formation may be treated by application of propane-2,2-diphosphonate.

Progress and prospects of ergot alkaloid research.

Ergot alkaloids, produced by the plant parasitic fungi Claviceps purpurea are important pharmaceuticals. The chemistry, biosynthesis, bioconversions, physiological controls, and biochemistry have been extensively reviewed by earlier authors. We present here the research done on the organic synthesis of the ergot alkaloids during the past two decades. Our aim is to apply this knowledge to the synthesis of novel synthons and thus obtain new molecules by directed biosynthesis. The synthesis of clavine alkaloids, lysergic acid derivatives, the use of tryptophan as the starting material, the chemistry of 1,3,4,5-tetrahydrobenzo[cd]indoles, and the structure activity relationships for ergot alkaloids have been discussed. Recent advances in the molecular biology and enzymology of the fungus are also mentioned. Application of oxygen vectors and mathematical modeling in the large scale production of the alkaloids are also discussed. Finally, the review gives an overview of the use of modern analytical methods such as capillary electrophoresis and two-dimensional fluorescence spectroscopy.

Application of oxygen vectors to Claviceps purpurea cultivation.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.

Free fatty acid and glucose metabolism during hypothermic perfusion of canine kidneys.

Canine kidneys were subjected to continuous nonpulsatile perfusion using 200 ml of a perfusate containing 50 g/l albumin. When optimal oxygenation was achieved, perfusate K+ contents were unchanged for 24 h, indicating adequate membrane function but tended to increase thereafter. Lowered oxygen pressures resulted in significant cellular K+ loss during the first hours of perfusion. During oxygenated perfusion, glucose and free fatty acids (FFA) were oxidized in considerable amounts with a preferential consumption of octanoate. A capacity for long-chain FFA oxidation became obvious when the octanoate had been used up, but the amount of these FFA in the perfusate depended preferentially on FFA being liberated from tissue lipids during the 1st day of perfusion. Glucose consumption rates were highest during the first 2 days of perfusion but the subsequent reduction of the metabolic rate was not accompanied by an accumulation of lactate. Thus medium-chain FFA and glucose should be supplied to the continuously perfused kidney in hypothermia and optimal oxygenation of the perfusate should be guaranteed. However, it seems to be unnecessary to supply exogenous long-chain FFA.

[Effect of prostaglandins on in vitro electrolyte metabolism in human spongiosa]. / Einfluss von Prostaglandinen auf den in-vitro-Elektrolytstoffwechsel menschlicher Spongiosa.

Prostaglandins seem to be involved in the pathogenesis of juxtaarticular osteopenia in rheumatoid arthritis. Therefore the influence of prostaglandins on in vitro electrolyte metabolism of human trabecular bone was tested. Trabecular bone was prepared from femoral heads of patients who had undergone hip replacement surgery for coxarthrosis. 500 mg samples of trabecular bone were incubated in modified Eagle's minimal essential medium. Net electrolyte movements between bone and incubation medium were measured. PGE2 caused an increase in the release of calcium (Ca) and magnesium (Mg) from bone into incubation medium as compared to controls (PGE2 1 micrograms/ml: Ca = 0.87 +/- 0.09 mmol/l*, Mg = 0.44 +/- 0.03 mmol/l*; controls: Ca = 0.81 +/- 0.09 mmol/l, Mg = 0.41 +/- 0.05 mmol/l; n = 15, *p less than 0.001). The effect of PGE2 was dose-dependent and comparable to the effect of human parathyroid hormone 1-34 (hPTH 1-34). PGE2 turned out to be the most potent prostaglandin on human trabecular bone. PGE1 and PGF2 alpha had about 50% and PGF1 alpha about 40% of the potency of PGE2. PGA1 and PGA2 had no effect. The effect of PGE2 could be completely inhibited by human calcitonin (hCT), similar to the inhibition of the effect of hPTH 1-34 by hCT. The effect of PGE2 was not further enhanced by hPTH 1-34. Magnesium metabolism was affected in all experiments in the same way as calcium metabolism. Phosphate metabolism, release of alkaline phosphatase and hydroxyproline from bone into incubation medium were not affected by prostaglandins.

Reconstructive surgery in the lower limb in hemophiliacs.

During the years 1973-76, we performed 76 operations on the lower limbs of hemophiliacs in an attempt to correct the disability due to intraarticular hemorrhages. The ways in which rehabilitative surgery can improve function and retard further damage are discussed in the light of the pathogenesis of disability due to intraarticular and intramuscular hemorrhage. Three particularly successful operative procedures are discussed: 1) supracondylar correction of knock-knee, 2) intertrochanteric correction of coxa valga, and 3) evacuation of iliacus hematoma in patients with paralysis of the femoral nerve.

Motion parallax: effects of blur, contrast, and field size in normal and low vision.

Can people with different forms of low vision use motion parallax to improve depth judgments? We used a staircase method to compare depth thresholds using motion parallax and static viewing. We tested eighteen normal-vision subjects with a range of simulated deficits in acuity, contrast sensitivity, and simulated peripheral-field loss, and ten low-vision subjects with a wide range of acuity, contrast sensitivity, and field loss. Subjects viewed three vertical cylinders monocularly and indicated which one was at a different depth from the other two. For motion-parallax trials, observers moved their heads (in a viewing assembly on rollers) from side to side over a range of 6-12 cm. For static trials, the viewing assembly was fixed in place. Normal-vision subjects' depth thresholds with motion parallax were significantly smaller than those with static viewing by an average factor of 1.95 (p < 0.05) across all levels of acuity and contrast. For low-vision observers, the depth thresholds exhibited large individual differences; however, the motion-parallax thresholds were smaller than the static thresholds by an average factor of 2.05 (p < 0.01). These findings indicate that motion parallax can provide useful depth information for people with low vision.

[Influence of fatty-acid and glucose content of the perfusate on the energy metabolism of the preserved canine kidney]. / Der Einfluss von Fettsäure- und Glucosegehalt des Perfusates auf den Energiestoffwechsel der konservierten Hundeniere.

Thirty-seven canine kidneys were preserved by hypothermic low flow perfusion for 24-120 h with solution I containing albumin, free fatty acids, and glucose, or with solution II containing Haemaccel without free fatty acids or glucose. Following 48 h preservation the renal ATP and SAN content was identical in both groups, irrespective of the availability of substrates from the perfusate.

Effect on myocardial metabolic pattern of local complete and incomplete ischemia.

After local complete ischemia at normothermia of 60, 100, 140, and 180 min duration the status of the adenylic acid-creatine phosphate system in the canine myocardium recovered to 98, 85, 74, and 30 percent of the control values, whereas glycogen was restored even more. In the infarcted myocardium the extent of alterations of the metabolic status was a function of the residual blood flow. Deviations from a regular metabolic status developed if the blood flow dropped below about 35 ml/min/100 gm. This critical flow rate is expected to vary with the energy requirement of the heart, but it is in keeping with results obtained by Rudolph and coworkers (personal communication) who found that patients with a myocardial blood flow below 30 ml/min/100 gm had a life expectancy of less than 1 month. In the nonaffected myocardium, both in experiments with local complete ischemia and in experiments with infarction, the metabolic status was always within normal ranges. This is in contrast to results published by Gudbjarnason (1971/1972) and Gudbjarnason, Puri, and Mathes (1971), who found that in noninfarcted myocardium tissue levels of ATP and creatine phosphate decreased to about 50 percent of the control values and that there was no restoration to normal values within 10 days after infarction.

[Effect of pO2 in perfusate on metabolism of preserved dog kidneys]. / Einfluss des pO2 im Perfusat auf den Stoffwechsel konservierter Hundenieren

In 48 canine kidneys submitted to an ischemia of 2 min duration normal cortical ATP values with a nearly unchanged content of total adenine nucleotides (SAN) were resumed by a five minute gravity perfusion. During the following 24 hours of low flow perfusion at 6 degrees C the extent of preservation of ATP and SAN correlated with the arterial oxygen pressure of the perfusate.