Protozoan, Bacterial, and Volatile Fatty Acid

Transcription

1 APPLIED MICROBIOLOGY, Nov. 1967, p Copyright 1967 American Society for Microbiology Protozoan, Bacterial, and Volatile Fatty Acid Changes Associated with Feeding Tylosin N. SATAPATHY1 AND D. B. PURSER' Institute of Nutrition and Food Technology, Ohio State University, Columbus, Ohio Received for publication 19 June 1967 Tylosin was fed to two of six wethers for 79 days, to a second two for only 28 days, and not at all to a third pair (controls). The addition of tylosin to the daily feed resulted in a rapid twofold increase in protozoal concentration and a change in the composition or characteristics, or both, of the bacterial population. The results indicate that the bacterial population was modified to the extent of about 80%. Total acid concentrations were initially depressed but appeared to be greater than those in control animals at the termination of the experiment. Deletion of tylosin from the ration resulted in a rapid decrease in protozoal concentrations, whereas changes in the bacterial population did not occur for a further 30 days. Previous reports from this laboratory (10, 13) have indicated that rumen protozoan concentrations may be modified by the inclusion of antibiotic in ruminant feeds. In one study (13), it was reported that either a residual effect or chlortetracycline-tylosin interaction exists. These results in part stimulated the investigation reported here. In addition, information concernmg changes occurring during prolonged antibiotic feeding was obtained. MATERIALS AND METHODS Sixrumen-fistulated wethers were randomly assigned to one of three pairs. The daily ration consisted of 700 g of basal pellets which contained alfalfa (40%), ground corn cobs (37.5%), shelled corn (15%), molasses (5%), and minerals (2.5%), together with 200 g of alfalfa meal. When fed, tylosin (15 mg/day) was mixed with the alfalfa meal. A seventh rumenfistulated animal was maintained on the control ration to supply standard inoculum for roll tubes and to supply rumen fluid for roll tube media. Tylosin was fed to one pair of animals for the entire experimental period (79 days), to the second pair for the first 28 days only, and not at all to the third pair (controls). Tylosin is weakly basic and appears to be a macrolide antibiotic similar to erythromycin, with the antibacterial spectrum being essentially gram-positive (9, 12). Rumen samples were collected before feeding (TO) and 4 hr post feeding (T4) on the 0, 1st, 2nd, 3rd, 9th, 1 Present address: Jawaharlal Neheyn Agricultural University, Jabalpur, India. 2Present address: Department of Animal Husbandry, Michigan State University, East Lansing, Mich th, 28th, 29th, 31st, and 37th day after the experiment had commenced, and thereafter every 7th day until the 79th day. Samples were collected as described previously (14). Volatile fatty acids (VFA) were quantitated by the method of Erwin et al. (7), protozoan concentrations were determined as described by Purser and Moir (14), and concentrations of viable bacteria were determined by the method of Bryant and Robinson (1) with the addition of 0.07% glycerol to the media. Tylosin concentrations were determined microbiologically on rumen fluid collected at T4 only; the analyses were carried out by Eli Lilly and Co. Viable bacteria were determined with two different systems. In the first (method A), individual medium was prepared with rumen fluid taken from each animal at T4 on each of the respective sample days; inoculum from the 7th or standard control sheep was used with each of these media. In the second system (method B), media were prepared containing rumen fluid from the 7th or standard control sheep, and tylosin was added to half of the tubes at a level calculated to approximate that found in rumen fluid of animals fed tylosin. Rumen fluid taken from each animal on each of the respective sampling days was used for inoculum. Quadruplicate tubes were used for each determination of viable bacteria. Individual sheep values for days 9, 28, 37, 58, and 79 are shown in each figure as the range from the treatment value. RESULTS Vol. 15, No. 6 Printed in U.S.A. Protozoan concentrations are shown in Fig. 1. The response to feeding tylosin was extremely rapid; thus, on the first day that tylosin was fed, the control animals at TO showed slightly greater concentrations of protozoa than the treated animals, whereas at T4 the treated animals showed

2 1418 SATAPATHY AND PURSER APPL. MICROBIOL. 0 TYLOSIN centrations were virtually identical with the con- 1 *s TYLOSN-COONTRO trol value. * In Fig.2b,results obtained with method B are..3/\0. presented as bacterial concentrations found with C-. the media plus tylosin as a percentage of con centrations obtained with the untreated media. Inoculum from control animals gave values of 11_2 oo lo about 20%, although a gradual increase in this Z* value over the experimental period appeared to ffi 'T. occur. On the other hand, 30% of the organisms z2 i (a) r \ in i F a 8 * \ inoculum from tylosin-fed animals grew on SM4 A media containing Tylosin by days 1 and 3, and --\ -A -. by day 9 a value of 90% had been reached. While highly variable, this high level was maintained a for the remainder of the experiment. A high level was also maintained for the second pair of ani-.w <t/mals, despite the removal of tylosin from their. 4 - ;. 4 o / feed on day 29; only by day 58 was there any 16 o indication of a decrease in this value. i120b b go in;e re OTYLOS:IN- L/ Xi\/ DAYS *TYLOSIN-ONTROL F FIG. 1. Protozoan concentrations in the rwnen of TJOLCOOL control sheep, sheep fed tylosin for 79 days, and sheep fed tylosin for only 28 days. Each value is the mean of X results from two animals. (a) Rwnen samples taken 4 hr after feed presentation (T4). (b) Runen samples taken prior to feed presentation (TO). 40 greater concentrations than the control animals. By day 1 at T4, an apparent plateau had been (a 4 reached, but this was not reached until day 3 at TO. Upon removal of tylosin from the ration on day 29, the protozoan concentrations decreased C rapidly, reaching a new level by day 31 at T4 -T / and by day 44 at TO. Concentrations in the con trol animals remained relatively stable throughout l the experimental period. Protozoan concen- t 0 trations at TO in animals continuously fed tylosin Q i increased a second time from day 51 until the I!, termination of the experiment. During this period, j concentrations rose from 1.8 x 106 organisms, A I20 to 2.3 X 106 organisms per ml. As in a previous experiment with this same ration (1 3), Entodinium K 0] p species comprised approximately 90% of the S L b- A total protozoan population. A A M0 In Fig. 2a, concentrations of viable bacteria, DAYS deternined according to method A, are presented FIG. 2. Viable rwnen bacteria concentrations from as the percentage of the values obtained for media control sheep, sheep fed tylosin for 79 days, and sheep containing rumen fluid from the control animals. fed tylosin for only 28 days. Each value is the mean of A marked reduction in the number of organisms results from two animals. (a) The inoculum was from growing in these media was apparent, reaching a seventh sheep, and the results from media containing b0 by 9, remaining rumen fluid from treated animals are expressed as a 50%byday 3 and 20% by day 9, and rpercentage of results from control animals. (b) The at this low level for the remainder of the experi- inocula were from the experimental animals, and the rement. The removal of tylosin on day 29 resulted sultsfrom mediaplus tylosin added in vitro are expressed in an immediate response; by day 31, the con- as a percentage of results from untreated media. NI

3 VOL. 15, 1967 EFFECT OF TYLOSIN ON RUMEN ORGANISMS 1419 Concentrations of viable bacteria for the individual animals as determined in the untreated media are given in Table 1. These values are considered to be too variable to allow any conclusions to be drawn with respect to the influence of tylosin on bacterial concentrations. Total VFA concentrations at TO are shown in Fig. 3, and are presented as total concentrations (Fig. 3b) and as percentages of the control values (Fig. 3a). After a transitory increase in total acid concentration, in the tylosin-fed sheep the concentration fell to 80 to 85 % of that of the control by day 9; it remained at this level until day 28. At this time, a marked increase in the acid concentration occurred, which resulted in an average value of 115% of that of the control for the remainder of the experiment. The deletion of tylosin from the feed on day 29 apparently forestalled such an effect for this treatment; a gradual rise in concentration occurred, and a level approximating that of the control was reached and maintained from day 51 to termination. Similar results were obtained with samples taken at T4. VFA proportions were determined and differences were noted but the results were considered to be too variable to be presented. Tylosin concentrations in rumen fluid taken from the animals are shown in Table 2. A steady concentration had been achieved in one pair of TABLE 1. Day Concentrations of viable bacteria in the rumen0 Treatment Continuous Tylosin 28 tylosin days only Control a Concentrations determined on untreated media including rumen fluid from a seventh animal. Numbers given should be multiplied times 108 to give the number of cells per gram. -A atylosin-control In CONTROL 4 1 1a} 0~~~~~~ 04 I~~ ~ ~ ~ ~ ~~~0 I E *-._ ; e so so DAYS FiG. 3. Total VFA concentration in the rumen of control sheep, sheep fed tylosin for 79 days, and sheep fed Tylosin for only 28 days. Each value is the mean of results from two animals. (a) Total acid concentration relative to concentration in the control animals as 100. (b) Total acid concentrations (pnoles/ml). animals (sheep 2 and 5) by day 3, but not in the other pair until the next sample. In the continuously fed animals, this level was maintained for the remainder of the experiment. Tylosin had disappeared from the rumen, in detectable quantities, with 2 days of deletion from the food. DIscussIoN Protozoan concentrations were markedly increased by the inclusion of tylosin in the feed. Concentrations during tylosin feeding were approximately double those for control animals, and the rapidity with which the response to tylosin feeding occurred was particularly striking. This effect is in complete agreement with previous results for both chlortetracycline and tylosin (13). It was hoped that the two systems used to determine bacterial concentrations would provide some insight into the magnitude of population modifications and further explain some of the results obtained previously (13). The inhibition of growth of bacteria, not previously exposed to tylosin, in media containing rumen fluid from

4 1420 SATAPATHY AND PURSER APPL. MICROBIOL. TABLE 2. Concentration of tylosin in rumen fluid taken from sheep 4 hr after feedinga Day Continuous tylosin pg/mi ,ug/mi Tylosin 28 days only pg/mi pug/ml a Analyzed by Eli Lilly and Co., Greenfield, Ind. tylosin-fed animals seems likely to be due to the residual tylosin in the rumen fluid taken from these animals. However, the influence of a changed microbial population on the ability of a rumen fluid to support bacterial growth (15) must still be considered as a possible factor. The high value on day 28 (53%) for animals removed from the tylosin treatment the following day cannot be explained, but it should be noted that the tylosin concentrations in the rumen appeared to be lower than those of the previous samples. Values in Fig. 2b provide an estimate of the ability of the bacterial population to grow in the presence of tylosin. That the bacterial population from animals fed tylosin was virtually 100% capable of growing in the presence of tylosin by day 9 was quite apparent, whereas only 20% of the organism from control animals could do so. Confirmation of this value of 20% is provided by the value of 20% of the organisms showing growth in media including rumen fluid from tylosin-fed animals (Fig. 2a). Whether the tylosin-resistant bacteria in the animals fed tylosin, i.e., virtually the entire population cultured from these animals, were the same as the tylosin-resistant bacteria in animals not exposed to tylosin, i.e., 20% of the total bacteria cultured from the animals, or included some bacteria which developed resistance after exposure to tylosin is not known. These results provide direct evidence to support the conclusion of previous workers (6, 8) that the depression and subsequent recovery of both appetite and digestibility, resulting from feeding antibiotics, were related to modifications within the microbial population. While it is now established that bacterial concentrations in the rumen behave inversely with respect to protozoan concentrations (2-5), differences in bacterial concentrations in the present experiments were not recorded. Indeed, in previous experiments with antibiotics (10, 13), differences in total viable bacterial concentrations have not been demonstrated either. A possible explanation is that a high degree of variability has been encountered with this parameter in work with antibiotics. An inverse relationship between bacterial and protozoan concentrations with defaunation work in this laboratory (11) has been demonstrated, and most of the previous workers (2, 5) obtained their results under similar conditions. The sequence of events following the inclusion of tylosin in rations fed to sheep appeared to be as follows. First, a marked and abrupt inhibition of bacterial activity (approximately 80% of the organisms appear to be susceptible to tylosin) occurred, lasting for 3 or more days, during which time protozoan concentrations doubled. This was accompanied by decreased total VFA concentrations. Second, a modification in the bacterial population occurred which was complete, at least with respect to composition, by the 9th day of tylosin feeding. Third, total VFA concentrations returned to those shown by control animals by day 29. Fourth, on the 58th day, protozoan concentrations appeared to show a further increase, and total acid concentrations tended to be greater than that of control animals. The rapid increase in protozoan concentrations in the first stage was probably the result of simple competition. This conclusion is supported by the decrease in total acid concentration and the fact that 80% of the original bacterial population was susceptible to tylosin. The reason for the second increase in protozoan concentration is not clear, and more work will be necessary both to elucidate and to confirm such an effect. Upon removal of tylosin from the animals' rations, the sequence of events was a rapid and marked decrease in protozoan concentrations and a gradual change in the bacterial population commencing 28 days after tylosin removal. This was shown by a decreased percentage of the population capable of growing in the presence of tylosin. Although this work leaves little doubt that the microbial population of the rumen was markedly modified when tylosin was included in rations fed to sheep, additional work is required to determine the significance of this modification with

5 VOL. 15, 1967 EFFECT OF TYLOSIN ON RUMEN ORGANISMS 1421 respect to overall rumen metabolism. It is also necessary to elucidate the nature of the modification of the bacterial population. ACKNOWLEDGMENT This investigation was supported by Eli Lilly and Co., Greenfield, Ind. LITERATURE CITED 1. BRYANT, M. P., AND I. M. ROBINSON An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44: BRYANT, M. P., AND N. SMALL The development of rumen microorganisms in inoculated vs. isolated growing calves. J. Dairy Sci. 39: BRYANT, M. P., AND N. SMALL Observa. tions on the ruminal microorganisms of isolated calves and inoculated calves. J. Dairy Sci. 43: EADIE, J. M The development of rumen microbial populations in lambs and calves under various conditions of management. J. Gen. Microbiol. 29: EADE, J. M., AND P. N. HOBSON Effect of the presence or absence of rumen ciliate protozoa on the total rumen bacterial count in lambs. Nature 193: EMERICK, R. J., AND L. B. EMERY Effect of chlortetracycline on methemoglobinemia resulting from the ingestion of sodium nitrate by ruminants. J. Animal Sci. 21: ERWIN, E. S., G. J. MARCO, AND E. M. EMERY Volatile fatty acid analyses of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44: EvANs, J. L., R. B. GRAINGER, AND C. M. THOMP- SON The effect of different levels and prolonged supplementation of chlortetracycline upon roughage digestion by sheep. J. Animal Sci. 16: HAMILL, R. L., M. E. HANEY, JR., M. STAMPER, AND P. F. WILEY Tylosin, a new antibiotic. II. Isolation properties and preparation of desmycosin, a microbiological active degradation product. Antiobiot. Chemotherapy 11: KLOPFENSTEIN, T. J., D. B. PURSER, AND W. J. TYZNIK Influence of Aureomycin on rumen metabolism. J. Animal Sci. 23: KLOPFENSTEIN, T. J., D. B. PURSER, AND W. J. TYZNIK Effects of defaunation on feed digestibility, rumen metabolism, and blood metabolites. J. Animal Sci. 25: MCGUIRE, W. S. BONICE, C. E. HIGGANS, M. M. HOEHN, W. M. STARK, J. WESTHEAD, AND R. N. WOLFE Tylosin, a new antibiotic. I. Microbiological studies. Antibiot. Chemotherapy 11: PURSER, D. B., T. J. KLOPFENSTEIN, AND J. H. CINE The influence of Tylosin and Aureomycin upon rumen metabolism and the microbial population. J. Animal Sci. 24: PURSER, D. B., AND R. J. MOIR Ruminal flora studies in the sheep. IX. The effect of ph on the ciliate population of the rumen "in vivo." Australian J. Agr. Res. 10: PURSER, D. B., AND R. B. TOMPKIN The influence of rumen fluid source upon viable bacteria counts and the cultivation of oligotrich and holotrich rumen protozoa. Life Sci. 4: