RESUMEN
An in vitro assay was used to examine biotransformation of toxic Senecio jacobaea pyrrolizidine alkaloids (PA) in ovine, bovine, and caprine rumen contents. Pyrrolizidine alkaloids were analysed by high performance liquid chromatography, and the rates of the alkaloid biotransformation were determined. The microbiological "Most Probable Numbers" technique was also used, in combination with thin-layer chromatography, to estimate relative numbers of rumen PA-biotransforming bacteria in the same samples. Pyrrolizidine alkaloids were biotransformed at average rates of 2.9 micrograms/ml/h (bovine), 25.6 micrograms/ml/h (caprine), and 19.2 micrograms/ml/h (ovine). Estimates of numbers of PA-biotransforming bacteria were 1.1 x 10(7) bacteria/ml rumen contents (bovine), 2.4 x 10(7) bacteria/ml (caprine), and 3.0 x 10(7) bacteria/ml (ovine). This project is among the first to quantitate rates of PA biotransformation in rumen contents and to identify caprine and bovine, in addition to ovine, rumen PA-biotransforming activity, as well as to estimate the actual numbers of PA-biotransforming bacteria in rumen contents.
Asunto(s)
Bacterias/metabolismo , Alcaloides de Pirrolicidina/metabolismo , Rumen/microbiología , Rumiantes/metabolismo , Animales , Biotransformación , Bovinos , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Femenino , Cabras/metabolismo , Técnicas In Vitro , Masculino , Ovinos/metabolismo , Especificidad de la EspecieRESUMEN
Ingestion of pyrrolizidine alkaloids, naturally occurring plant toxins, causes illness and death in a number of animal species. Senecio jacobaea pyrrolizidine alkaloids cause significant economic losses due to livestock poisoning, particularly in the Pacific Northwest. Some sheep are resistant to pyrrolizidine alkaloid poisoning, because ovine ruminal biotransformation detoxifies free pyrrolizidine alkaloids in digesta. Antibacterial agents modify ruminal fermentation. Pretreatment with antibacterial agents may account for some animal variability in resistance to pyrrolizidine alkaloid toxicosis, and antibacterial agents can also be used for characterizing ruminal pyrrolizidine alkaloid-biotransforming microflora. The objective of this study was to evaluate the effects of antibacterial agents on biotransformation of a predominant S. jacobaea pyrrolizidine alkaloid, jacobine, in ovine ruminal contents. Ovine ruminal jacobine biotransformation was tested in vitro with 20 independent antibacterial agents. Low amounts of rifampin and erythromycin prevented jacobine biotransformation. Chlortetracycline, lasalocid, monensin, penicillin G, and tetracycline were slightly less effective at inhibiting jacobine biotransformation. Bacitracin, crystal violet, kanamycin, and neomycin were moderately inhibitory against jacobine biotransformation. Brilliant green, chloramphenicol, gramicidin, nalidixic acid, polymyxin B SO4, sodium azide, streptomycin, sulfisoxazole, and vancomycin had little to no effect on jacobine biotransformation. The antibiotics that were most effective at inhibiting biotransformation were those that are active against gram-positive bacteria. Therefore, gram-positive bacteria are most likely critical members of the jacobine-biotransforming consortia.
Asunto(s)
Antibacterianos/farmacología , Alcaloides de Pirrolicidina/metabolismo , Rumen/efectos de los fármacos , Animales , Biotransformación/efectos de los fármacos , Femenino , Técnicas In Vitro , Hígado/efectos de los fármacos , Alcaloides de Pirrolicidina/toxicidad , Rumen/metabolismo , Rumen/microbiología , OvinosRESUMEN
Anaerobic production of extracellular polysaccharide (EP) was examined, using a previously uncharacterized, obligately anaerobic rumen isolate, Butyrivibrio fibrisolvens nyx, which produced an EP that was rheologically similar to xanthan gum. The main objectives were to determine the nutritional requirements and conditions which promoted EP production by strain nyx. Strain nyx was grown anaerobically in defined and semidefined media. In addition to carbohydrate and nitrogen sources, strain nyx required acetic acid, folic acid, biotin, and pyridoxamine. Strain nyx produced similar amounts of EP at 35 to 40 degrees C. Conditions that improved growth usually improved EP production. Of the carbohydrates tested, glucose supported the fastest growth and most EP production, followed by sucrose, xylose, and lactose. Strain nyx utilized ammonium sulfate, urea, or vitamin-free casein hydrolysate as nitrogen sources for growth and EP production. At 2 and 20 g/liter, respectively, ammonium sulfate and vitamin-free casein hydrolysate supported about the same rates of growth and EP production. EP was not produced in the lag or stationary phases, and EP production was exponential during exponential cell growth. Based on the results of this work, anaerobic EP production with B. fibrisolvens nyx could reduce energy costs for industrial EP production compared with the cost of aerated systems. Finally, this work demonstrated that, under appropriate growth conditions, a gastrointestinal tract (ruminal) microorganism produced high levels of EP.
RESUMEN
Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39 degrees C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST(50)) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST(50) = 6.6 h at a dilution rate of 0.33 h) than for suspensions that had been limited by nitrogen (ST(50) = 9.5 h at a dilution rate of 0.33 h). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST(50) = 7.1 h) than for nitrogen-limited cells (mean ST(50) = 12 h). Although it has relatively low ST(50) values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.
RESUMEN
Titanium citrate (TC) or L-cysteine-sodium sulfide was added as a reducing agent to buffers and agar media used for enumeration of bacteria from rumen contents of high-forage-fed steers. Approximately equal colony counts were found on TC and L-cysteine-sodium sulfide-reduced media with rumen contents taken 8 h postfeeding, when active bacterial growth was occurring. The colony counts on TC medium were only 56% of those with L-cysteine-sodium sulfide medium with rumen contents taken 1 h prefeeding when bacterial growth was minimal. When colonies from L-cysteine-sodium sulfide medium were transferred to TC medium and vice versa, almost all colonies grew. The data indicate that TC can be inhibitory to bacteria upon their initial isolation from natural habitats, particularly when growth rates are low in these habitats.