Cytotoxic effect of aminoglycoside antibiotics on the mammalian cell lines.

Aminoglycoside antibiotics have been used for treating serious but also routine infections in veterinary and human medicine for many years. The basic aim of this work is to evaluate the cytotoxicity of dihydrostreptomycin and neomycin in vitro on three cell cultures - BHK-21 (Syrian golden hamster kidney fibroblast), VERO (African green monkey kidney fibroblast) and FEA (feline embryonic fibroblast) cells. The morphological changes were examined by Giemsa staining. Cells were dried and visualized under fluorescence microscope. After the exposure to different experimental doses of dihydrostreptomycin (812.5-20000 µg/mL) and neomycin (1000-20000 µg/mL) during 24 h, the viability of BHK-21, FEA and VERO cell lines were evaluated by MTT assay. Viability of BHK-21 cells significantly (P < 0.001) decreased after treatment with 3500; 5500 and 7500 µg/mL of dihydrostreptomycin and 9000; 10000 and 20000 µg/mL of neomycin. The FEA cell viability decreased significantly (P < 0.001; P < 0.01) at 2500 and 3000 µg/mL dihydrostreptomycin and at 3000 µg/mL of neomycin treatment. Only the highest concentration of dihydrostreptomycin (20000 µg/mL) reduced VERO cell viability significantly (P < 0.01). Based on or results we can assume the effect of different antibiotics in different concentrations on cell lines is various. Detection of antibiotic toxicity to animal cells is very important because of the increasing resistance of bacteria. One of the solutions is drug dose increasing, but only to a certain concentration, since the toxic effect over the therapeutic one will prevail, which we have also shown in this work.

Events occurring during the previous lactation, the dry period, and peripartum as risk factors for early lactation mastitis in cows receiving 2 different intramammary dry cow therapies.

The objective of this study was to investigate the association between mastitis events occurring during the previous lactation, the dry period, and the peripartum period on the incidence of early lactation mastitis in cows receiving ceftiofur hydrochloride or penicillin dihydrostreptomycin as intramammary dry cow antibiotic therapy. Cows (n=402) from 2 large dairy farms in Central Florida were enrolled in the study at the time of dry-off processing and were randomly assigned to 1 of 2 dry cow therapies: ceftiofur hydrochloride or penicillin dihydrostreptomycin. Composite milk samples were collected at dry-off and after calving for bacteriological examination and somatic cell count. Peripartal health disorders were monitored during the first 30 d of lactation and included calving difficulty, metritis, ketosis, and left displaced abomasum. Milk production and individual somatic cell scores (SCS) were recorded monthly by the Dairy Herd Improvement Association. The main outcome variables were the risk of clinical mastitis during the first 30 and 60 d of lactation, and the risk of subclinical mastitis at the first 2 monthly Dairy Herd Improvement Association tests after calving (up to 70 d in milk). Additionally, the SCS and the presence of mastitis pathogens in milk at dry-off and at calving were analyzed. Explanatory variables consisted of events occurring during the previous lactation, at dry-off and during the dry period, at calving, and within the first 30 d after calving. Multiple events occurring during the previous lactation had a significant effect on the incidence of mastitis in the subsequent lactation. These events included low milk yield, intermediate lactation length, clinical mastitis, and lactation SCS average. Similarly, intramammary infections with environmental bacteria at dry-off increased the chances of clinical mastitis the first month after calving. Dry-off therapy had a significant effect on mastitis incidence; cows treated with ceftiofur hydrochloride had lower odds of having clinical and subclinical mastitis in the subsequent early lactation compared with cows treated with penicillin dihydrostreptomycin.

Streptococcus dysgalactiae isolates at calving and lactation performance within the same lactation.

The objective of the study was to investigate the association between early lactation Streptococcus dysgalactiae isolates and milk yield, somatic cell count (SCC), clinical mastitis, and culling in the same lactation. The 178 commercial dairy herds were randomly placed into 3 penicillin- or penicillin-dihydrostreptomycin-based dry-cow treatments and 3 different postmilking teat disinfection groups-negative control, iodine, or external teat sealant. All cows were sampled in early lactation, and Strep. dysgalactiae-positive and culture-negative cows were followed throughout the remainder of the lactation. Mixed models, including repeated measurements, with test-day observation as dependent variable, were used to compare milk yield, SCC, and available milk quality variables throughout the remaining lactation. Survival analyses, using a positive frailty model to account for any herd random effects, were used to estimate the hazard ratio for clinical mastitis and culling. Streptococcus dysgalactiae-positive cows had a significantly higher SCC throughout the lactation compared to culture-negative cows. For primiparous or multiparous cows, respectively, the differences in the geometric mean SCC between Strep. dysgalactiae-positive and culture-negative cows was 197,000 or 280,000 cells/mL at the beginning of the lactation, 24,000 or 46,000 cells/mL in mid lactation, and 39,000 or 111,000 cells/mL at the end of the lactation. Streptococcus dysgalactiae-positive primiparous or multiparous cows produced 334 or 246 kg less milk, respectively, during a 305-d lactation compared with culture-negative cows. Compared with culture-negative cows, the hazard ratios for clinical mastitis in Strep. dysgalactiae-positive cows were 2.3 (1.9 to 2.9) and 1.6 (1.3 to 2.0) for culling. For cows with both Strep. dysgalactiae and Staphylococcus aureus isolates, the hazard ratio for culling significantly increased to 2.5 (1.9 to 3.2).

Clinical mastitis in Norwegian herds after a combined selective dry-cow therapy and teat-dipping trial.

The objective of this study was to see if introduction of a 2-yr combined selective dry-cow therapy and teat-dipping trial would reduce clinical mastitis (CM) events in 164 Norwegian dairy herds. Three different penicillin or penicillin/dihydrostreptomycin-based dry-cow treatments, and 3 different teat-dipping regimens (negative control, iodine teat dip, or an external teat sealant) were independently and randomly allocated to each herd. Complete lactations both before and during the trial were investigated. Altogether, 1,005 CM cases were recorded in the lactations before the trial and 924 cases were recorded during the trial. Bacteriological milk samples were available from 784 of the 924 CM cases during the trial. Among these, Staphylococcus aureus were isolated from 47.4%, Streptococcus dysgalactiae from 22.5%, Escherichia coli from 10.7%, and coagulase-negative staphylococci from 6.3%. In addition, 12.5% cases were bacteriological negative, and the remainder of the CM cases were caused by other microbes. The different models were analyzed using Cox regression analysis with PROC PHREG and a positive stable frailty model in the SAS macro. Separate models were made for cows housed in tie-stalls and free-stalls. Parity had a significant impact on the CM risk in both type of stalls. Older cows (parity > 3) had the highest hazard ratio of contracting CM in tie-stalls (1.68) and free-stalls (2.18) compared with parity 1. The CM risk decreased significantly (13%) in tie-stalls and by 18% in free-stalls. In tie-stalls, iodine-dipped cows had a significantly lower chance (21%) of getting CM compared with the negative control and the use of external teat sealant. The same trend was seen in free-stalls; however, the differences were not significant. Compared with CM before the trial, the reduction of CM was 15% during the trial.

The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels.

The most serious side-effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular dihydrostreptomycin not only blocks the mechano-electrical transducer channels of mouse outer hair cells at negative membrane potentials, as previously shown, but also enters the cells through these channels, which are located in the cells' mechanosensory hair bundles. The voltage-dependent blocking kinetics indicate an open-channel block mechanism, which can be well described by a two barrier-one binding site model, quantifying the antibiotic's block of the channel as well as its permeation in terms of the associated rate constants. The results identify the open transducer channels as the main route for aminoglycoside entry. Intracellularly applied dihydrostreptomycin also blocks the transducer channels, but at positive membrane potentials. However, the potency of the block was two orders of magnitude lower than that due to extracellular dihydrostreptomycin. Extracellular Ca2+ increases the free energy of the barrier nearest the extracellular side and of the binding site for dihydrostreptomycin. This reduces both the entry of dihydrostreptomycin into the channel and the channel's affinity for the drug. In vivo, where the extracellular Ca2+ concentration in the endolymph surrounding the hair bundles is < 100 microM, we predict that some 9000 dihydrostreptomycin molecules per second enter each hair cell at therapeutic drug concentrations.

An unusual presentation of suspected oedema disease of swine in Kenya.

From a group of 11 recently weaned pigs, 4 were reported to be sick. Clinical examination of the sick pigs revealed marked dyspnoea, bluish-red discolouration of the skin, incoordination and difficulty in walking. Bacteriological examination of the gut contents of 2 pigs that had died earlier yielded pure cultures of haemolytic Escherichia coli. Post mortem examination of the remaining 2 pigs that died subsequently revealed progressive pulmonary collapse. One of these also showed subcutaneous oedema of the head and marked oedema of the mesentery of the spiral colon and oedema of the brain. Microscopically there was pulmonary alveolar collapse and degenerative changes in the liver. On the basis of the clinical signs, isolation of haemolytic E. coli and the post mortem findings, a diagnosis of oedema disease was made.

Amoxycillin as an alternative to dihydrostreptomycin sulphate for treating cattle infected with Leptospira borgpetersenii serovar hardjo.

OBJECTIVE: To assess the effect of amoxycillin treatment on urinary excretion of leptospires from cattle infected with Leptospira borgpetersenii serovar hardjo. DESIGN: A chemotherapy trial with controls. PROCEDURE: Fourteen heifers serologically negative to L hardjo were inoculated with L hardjo via the conjunctival route and assessed for evidence of infection by serological, fluorescent antibody and microbiological tests. Two injections (48 h apart) of amoxycillin at a dose of 15 mg/kg were administered intramuscularly to seven heifers 6.5 weeks after infection; the remaining heifers acted as untreated controls. Later, these seven control group heifers were treated with a single dose of amoxycillin (15 mg/kg). Samples of urine were collected before and after amoxycillin treatments; kidneys were collected at slaughter, and examined by fluorescent antibody test and microbiological culture. RESULTS: Leptospires were isolated from the urine of 11 of 14 heifers inoculated with L hardjo. After treatment of six of these with two injections of amoxycillin, leptospires were not isolated. Of the controls, four of the five initially leptospiruric heifers continued to shed leptospires; after a single injection of amoxycillin, no leptospires were detected in the kidneys of these four. CONCLUSION: Amoxycillin may be an acceptable alternative to dihydrostreptomycin sulphate for the treatment of cattle infected with L hardjo.

Comparison of two regimens for the treatment of clinical bovine mastitis caused by bacteria sensitive to penicillin.

The efficacies of two regimens for the treatment of acute clinical mastitis were compared in a randomised multi-centre field trial in Norway, using 657 cows. The purpose was to determine whether repeated intramuscular injections of penicillin G for three days were more effective than a single injection, when given in combination with intramammary treatment for five days. The results were evaluated on the basis of clinical and microbiological examinations and cell count determinations of quarter milk samples taken at the initial visit and four weeks later. There were no significant differences between the effects of the treatments, either for all the cows, or for subgroups of the cows based on age, stage of lactation, and systemic reaction, or the type of causal bacteria.

Bioavailability in the rabbit of penicillin and dihydrostreptomycin from three commercial penicillin/aminoglycoside fixed combination products for intramuscular injection.

The bioavailability of penicillin and dihydrostreptomycin from three penicillin/ aminoglycoside fixed combination products for intramuscular injection was investigated in a four-way, randomized, crossover experiment in rabbits. Attention is focused on bioequivalence based on plasma concentration vs. time profiles to study whether the rabbit is a good model to detect differences in in vivo delivery of penicillin and/or dihydrostreptomycin after intramuscular administration of different products. In all products, penicillin was present as a suspension. Although the extent of absorption of penicillin did not differ between the three products, large differences in the rates of absorption were observed. With respect to dihydrostreptomycin, no significant differences were observed between the products. The results from this study demonstrate that the rabbit is a good model to detect differences in bioavailability of suspended penicillin from penicillin /dihydrostreptomycin fixed combination products for intramuscular injection. A study with the same products is presently being carried out in calves to investigate whether bioequivalence studies in rabbits could replace studies in the target animals.

Bioequivalence study in calves of three commercial penicillin/dihydrostreptomycin fixed combination products for intramuscular injection.

A bioequivalence study with three penicillin/dihydrostreptomycin fixed combination products for intramuscular administration was performed in dairy calves. In addition to plasma concentrations of penicillin and dihydrostreptomycin, creatine phosphokinase concentrations were determined during a period of 72 h after administration of the drug products. Considerable differences were observed in the pharmacokinetics of penicillin from the three products. Although the extent of absorption was similar for all products, one product showed a significantly slower release from the site of injection. Except for the AUC, the 90% confidence intervals for these parameters exceeded the acceptable range of 0.80-1.20. Therefore, these products are not bioequivalent with respect to the rate of absorption of penicillin. Concerning the pharmacokinetics of dihydrostreptomycin in calves, it could not be concluded that the products were bioequivalent, since the 90% confidence intervals of the ratios for Cmax, tmax and MRT exceeded the range of 0.80-1.20. From this study in calves, it was also found that the product with the slowest release of penicillin from the injection site caused the most severe tissue damage, based on plasma creatine phosphokinase concentrations. Comparing the results from this study in calves with those from a previous study in rabbits, it can be concluded that the rabbit is a good animal model that could substitute for large animals, at least calves, in bioequivalence studies for penicillin/dihydrostreptomycin fixed combination products.

Effect of dry-cow therapy on subclinical mastitis--an evaluation of long-acting and short-acting intramammaria.

This field study was conducted to evaluate the effect of selective dry-cow therapy with long-acting and short-acting antibiotics, respectively, and also in comparison to control groups without antibiotic treatment. A total of 684 cows from 288 different herds in three Norwegian regions fulfilled the criteria of the study design. There were 104 cows in control group A (sampling only), 115 cows in control group B (placebo), 221 cows treated with long-acting intramammaria Benestermycin vet. 'Leo' for 1 day at drying off in group C, and 244 cows treated with four short-acting intramammaria Leocillin with Dihydrostreptomycin vet. 'Leo' every second day before drying off in group D. The overall effect, measured as the cow being healthy after therapy, was 14.2% in control groups and 33.7% in therapy groups 30 +/- 17 days into the next lactation. Of quarters infected with S. aureus both in late lactation (45 +/- 32 days before drying off) and at drying off, 38.4% in the control group were bacteriologically negative 30 +/- 17 days into the next lactation, compared with 49.5% in the long-acting group and 68.6% in the short-acting group. Of quarters infected with Str. dysgalactiae both in late lactation (45 +/- 32 days before drying off) and at drying off, 10 out of 27 were still infected with Str. dysgalactiae in the control group 30 +/- 17 days into next lactation, compared with 0 out of 31 in the therapy groups. Dry-cow therapy in coagulase-negative Staphylococcus spp. (CNS)-infected quarters led to a 5.2 odds ratio of being healthy quarters 30 +/- 17 days into the next lactation, compared with control groups. Despite this, the overall frequency of CNS in the material was unchanged after therapy compared with controls. Short-acting compared to long-acting preparations had a significantly better effect in preventing new infection with S. aureus or Str. dysgalactiae in untreated healthy quarters in cows with fewer than three infected quarters. This difference in preventive effect was greater in cows with one infected quarter during previous lactation (the new infection rates being 0.078 for short-acting and 0.149 for long-acting) than in those with two infected quarters (the new infection rates being 0.042 and 0.063, respectively).

Successful treatment of mycoplasmosis in layer chickens with single dose therapy.

The efficacy of treatment with single dose administration of 5 drugs at different dosages to layer hens naturally infected with Mycoplasma gallisepticum was studied. The drugs were tiamulin, which was administered orally, tylosin (parenterally and orally), spiramycin (orally), long-acting oxytetracycline (parenterally) and tylosindihydrostreptomycin (parenterally). Cure was assessed by the absence of nasal discharge. The cure rate was significantly higher (P less than 0.05) in treated hens than in untreated hens, as early as 1 day after treatment. Remission for 33 days was achieved in 60% of hens treated with 100 mg oxytetracycline, in 100% of hens treated with 100 mg or 200 mg spiramycin, in 92% and 85% of hens treated with 100 mg tylosin, parenterally and orally, and in 89% and 88% of birds given 100 mg tiamulin and tylosin-dihydrostreptomycin, respectively.

[The pharmacokinetics of dihydrostreptomycin sulfate in domestic pigeons (Columba livia Gmel., 1789 var. domestica)]. / Zur Pharmakokinetik von Dihydrostreptomycinsulfat bei Haustauben (Columba livia Gmel., 1789 var. domestica).

A 20% aqueous solution of dihydrostreptomycin sulfate in a dosage of 100 mg/kg b.w. is non-toxic for pigeons, although renal accumulation occurs. 12 hours after a single subcutaneous injection of 100 mg/kg b.w. the plasma concentration decreases to 3.64 micrograms/ml and therefore below the therapeutically effective concentration, while in birds suffering from salmonella infections plasma levels decrease to comparative concentrations (3.84 micrograms/ml) after only 8 hours. Contrary to the directions for use in pigeons of the preparation CX 60, the subsequent dosage should be given no later than 10 hours after the first application.

Residues of aminoglycoside antibiotics in eggs after medication of laying hens.

1. The transfer of aminoglycoside antibiotics into eggs was determined separately from albumen, yolk or whole egg after oral administration of dihydrostreptomycin (DHS), neomycin and spectinomycin and after an intramuscular injection of DHS. Residues were assayed by an agar plate diffusion method in cylinders with a specific test organism for each antibiotic. 2. Only DHS, administered by the intramuscular route, led to detectable residues in eggs. The total amount of DHS excreted via the eggs represented 1% of the dose administered. 3. Residues in the whole egg were detected for 8 d.

Clinical trial of three therapeutic regimens for bovine mastitis.

An open, block randomised multi-centre clinical trial was performed in Norway during 1985 to 1987 to compare the therapeutic efficacy of three antibiotic regimens against clinical bovine mastitis caused by penicillin-sensitive bacteria. Two regimens consisted of procaine penicillin injected intramuscularly for either three or five days, and the third, the traditional Norwegian regimen, consisted of one intramuscular injection of a combination of procaine penicillin and dihydrostreptomycin followed by one intramammary treatment daily per infected quarter for four days. The study included 621 quarters with infectious mastitis from 439 cows. The most efficient regimen for all bacteria was five days systemic treatment (53.1 per cent cured), and the traditional regimen was second best (46.7 per cent cured). The least efficient regimen consisted of systemic therapy with procaine penicillin for three days (36.9 per cent cured). The difference between the therapeutic efficacies of the three regimens was reduced when the clinical mastitis was severe, and in severe mastitis caused by Staphylococcus aureus the difference was very small.

Antibiotic levels in bovine lacrimal fluid after single application of ointments containing procaine benzyl penicillin plus dihydrostreptomycin; and benzathine cloxacillin.

The efficacy of an experimental slow-release formulation, containing procaine benzyl penicillin and dihydrostreptomycin, was investigated in a cross-over study with a cloxacillin eye ointment in 12 cows with clinically normal eyes. After a single topical application the therapeutic concentrations of penicillin were sustained for 48 to 92 hours and of cloxacillin for 32 to 48 hours. These long-acting ointments will simplify the successful treatment of painful eye disorders such as keratoconjunctivitis. A practical and non-irritant method for sampling tears is described.