Influence of flavomycin on ruminal fermentation and microbial populations in sheep

Joan E. Edwards{dagger}, Neil R. McEwan, Nest McKain, Nicola Walker{ddagger} and R. John Wallace

Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK

Correspondence
R. John Wallace
rjw{at}rri.sari.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Flavomycin is a phosphoglycolipid antibiotic that promotes growth in ruminants. The aim of this study was to characterize the effects of flavomycin on ruminal micro-organisms and their metabolic consequences. In sheep receiving a mixed grass hay/concentrate diet, inclusion of 20 mg flavomycin day–1 decreased ruminal ammonia and total volatile fatty acid concentrations (P<0·001), but the acetate : propionate ratio was unchanged. Ruminal pH tended to be lower with flavomycin, and ammonia-production rates of ruminal digesta from control animals measured in vitro tended to be inhibited by flavomycin. Pure-culture studies indicated that anaerobic fungi, protozoa and most bacterial species were insensitive to flavomycin. Fusobacterium necrophorum was the most sensitive species tested, along with some high-activity ammonia-producing (HAP) species. Effects on F. necrophorum in vivo were inconsistent due to large inter-animal variation. HAP numbers appeared to be decreased. Changes in the rumen bacterial-community structure were assessed by using denaturing-gradient gel electrophoresis (DGGE) analysis of rumen digesta 16S rRNA. DGGE profiles differed from animal to animal, but remained consistent from day to day. The community structure changed when flavomycin was introduced. The roles of F. necrophorum and HAP species in ammonia formation and of F. necrophorum in the invasion of wall tissue are consistent with the observed effects of flavomycin on ruminal ammonia formation and, in other studies, on decreasing tissue-turnover rates.


Abbreviations: DGGE, denaturing-gradient gel electrophoresis; HAP, high-activity ammonia-producing; PCH, pancreatic casein hydrolysate; VFA, volatile fatty acids

{dagger}Present address: Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK.

{ddagger}Present address: AgResearch, Private Bag, Palmerston North, New Zealand.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Flavomycin is a phosphoglycolipid antibiotic that has been used exclusively as an antimicrobial growth promoter since its discovery in the mid-1950s (Wallhausser et al., 1965; Bauer & Dost, 1971). It has been used most extensively in pig- and poultry-production systems, but flavomycin also promotes growth in ruminants and a variety of other species (Rebolini et al., 1982). Flavomycin inhibits bacterial growth by competitive inhibition of the enzyme that catalyses the transglycosylation reaction during peptidoglycan biosynthesis (Van Heijenoort et al., 1978; Huber, 1979; van Heijenoort, 2001). However, the growth-promotion mechanism of flavomycin, particularly in ruminants, is unclear. Its mode of action on the rumen microbial population appears to differ from that of the well-characterized ionophore class of antimicrobials (Febel et al., 1988) in that volatile fatty acid (VFA) proportions are generally unchanged (Cafantaris, 1981; Rowe et al., 1982; Galbraith et al., 1983; Ahrens, 1987; Febel et al., 1988; Masoero et al., 1991; Edwards et al., 2005). Protein metabolism seems to be affected, not only in the rumen (Cafantaris, 1981; Van Nevel & Demeyer, 1987), but also in the lower part of the digestive tract (Edwards et al., 2005). In addition, flavomycin appears to decrease the turnover rate of gut-wall tissues (MacRae et al., 1999; Edwards et al., 2005). The reasons for these responses, in terms of ruminal species sensitive to flavomycin, are not known.

Growth-promoting antibiotics, including flavomycin, are being phased out of animal production in Europe in anticipation of a total ban on their use at the end of 2005. It is important to discover how they work, in order that other, non-hazardous means can be devised to deliver a similar mode of action on the gut microbial community and to provide similar production benefits. The aim of the present study was to characterize the antimicrobial effects and metabolic consequences of flavomycin in the ruminal ecosystem. Some of these results have been presented previously in a preliminary form (McKain et al., 2000; Wallace et al., 2001; Edwards et al., 2002).


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Animals and sampling.
Experiments with ruminally cannulated sheep were carried out as a regulated procedure with Home Office permission. Four adult ruminally cannulated sheep received a maintenance diet [500 g hay, 299·5 g barley, 100 g molasses, 91 g fishmeal and 9·5 g vitamins and minerals (kg dry matter)–1], fed twice daily. Before (–1 and 0 days) and after 7, 8, 14 and 15 days of daily flavomycin supplementation (250 mg Flavomycin80, which equals 20 mg flavomycin day–1; SCA Nutrition), ruminal contents were sampled 0, 1, 2, 4 and 6 h after morning feeding. The pH was measured immediately and samples were then processed for analysis of fermentation characteristics. On days –1, 0, 14 and 15, the 2 h samples were also used to enumerate viable bacterial populations [total viable anaerobes, Fusobacterium necrophorum and high-activity ammonia-producing (HAP) species]. Ruminal contents were also collected daily (2 h after morning feeding) throughout the experimental period and stored separately at –20 °C for later DNA extraction and analysis by denaturing-gradient gel electrophoresis (DGGE).

In an experiment to determine the effect of flavomycin on bacterial numbers in ruminal digesta and associated with the ruminal wall, 14 male, 6-month-old, 30 kg lambs were divided into two groups that were matched for weight. Both groups were fed the same grass hay/concentrate diet as before for 6 weeks. For the last 2 weeks of this period, one group received 250 mg Flavomycin80 day–1. At the end of the 6-week period, animals were killed by lethal injection. Tissue samples were dissected from the dorsal sac of the rumen, then were placed into sterile universal bottles flushed with CO2. The universal bottles for ruminal wall-tissue collection contained 10 ml defined basal anaerobic diluting medium (Wallace, 1978). Samples were transferred under CO2 to the laboratory for processing and analysis.

The ruminal wall-tissue samples were taken into an anaerobic chamber and washed vigorously three times in 10 ml defined basal anaerobic diluting medium in order remove loosely attached bacteria. The keratinized papillae were cut away from the muscle layer of the washed tissue section by using a sterile scalpel and suspended in a pre-weighed universal bottle containing 10 ml defined basal anaerobic diluting medium. The papillae suspension was then homogenized (Ultra-Turrax T25 tissue homogenizer) for 1 min at 20 000 r.p.m. under CO2.

Samples of ruminal digesta (0·5 g) or homogenized rumen-wall papillae (0·5 ml) were used to prepare a tenfold dilution series (D1–D10) in defined basal anaerobic diluting medium. The serial dilutions were used to inoculate microtitre plates for the enumeration of total viable bacteria and F. necrophorum (below). The number of bacterial cells was calculated [(g wet digesta)–1 or (g rumen wall papillae)–1].

Analytical methods.
Ruminal digesta samples were analysed for VFA content by GC as described previously (Newbold et al., 1995). Ruminal concentrations of L-lactate were assayed enzymically (Hochella & Weinhouse, 1965) and ammonia was determined by using an adaptation of the phenol/hypochlorite method (Whitehead et al., 1967).

Ammonia-production rates.
The influence of flavomycin on ammonia production was determined in ruminal digesta in vitro and compared with the influence of the ionophore monensin. Samples of ruminal digesta were removed via the ruminal cannula from four sheep on the maintenance diet 3 h after feeding. Samples were kept warm and under CO2, and strained through four layers of muslin. Ammonia-production rates were measured by two different methods. The first method was intended to determine the rate of ammonia production from endogenous protein in ruminal digesta; the second method was that used by Russell et al. (1988), in which ruminal fluid is diluted fourfold in basal growth medium and incubated for 6 h with an exogenous protein source [pancreatic casein hydrolysate (PCH)]. The latter method is referred to as the ‘batch-culture’ type of incubation. In the first method, triplicate incubations were made, at 39 °C and under CO2, with 50 ml strained ruminal fluid, to which was added 16 µl ethanol, 16 µl 6·25 mg flavomycin ml–1 in ethanol (giving a final flavomycin concentration of 2 µg ml–1) or 16 µl monensin (10 mg ml–1) in ethanol. Samples (4 ml) were removed at 0, 0·5, 1, 2 and 4 h into 1·0 ml 30 % perchloric acid. The mixture was chilled and then centrifuged (15 000 g, 15 min, 4 °C) and ammonia concentration was measured in the supernatant. The ammonia-production rate was calculated by linear regression. Triplicate incubations were carried out in the batch-culture incubation, carried out as described by Russell et al. (1988), with the same additions as were carried out in the first method. Ammonia and protein concentrations were measured at 0 and 6 h. The specific rate of ammonia production was calculated from these values by using the mean of protein concentrations at 0 and 6 h.

Ammonia-production rates of pure cultures were determined by the method of Russell et al. (1988), using bacteria grown in Hobson's M2 liquid medium (Hobson, 1969) or in the defined medium of Russell et al. (1988), which contains 15 g PCH l–1.

Micro-organisms.
The bacteria used in this study are held in the Rowett Research Institute (RRI) culture collection or were obtained from the American Type Culture Collection (ATCC), German Collection of Microorganisms and Cell Cultures (DSMZ), Japanese Collection of Microorganisms (JCM) or were kindly gifted (see Tables 1 and 2). The ruminal fungal species (Neocallimastix frontalis RE1, Neocallimastix patriciarum Cx and Piromyces sp. P) and archaeon (Methanobrevibacter smithii PS) used in this study were grown from stock cultures held in the RRI culture collection. Mixed ruminal protozoa samples were obtained from ruminally cannulated sheep receiving a maintenance diet as described above.


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Table 1. Sensitivity of different species of ruminal bacteria to flavomycin

 

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Table 2. Sensitivity of Fusobacterium species to flavomycin and their ammonia-production activity when grown on different media

 
In vitro sensitivity of micro-organisms to flavomycin.
A flavomycin reference standard, kindly donated by Hoechst AG, Frankfurt, Germany, was used for in vitro studies. The sensitivity of bacteria to flavomycin was assessed by using an MIC method. Sensitivity was defined as the lowest concentration of flavomycin that resulted in the decrease of bacterial growth by 50 or 90 % (MIC50 or MIC90). Cultures were inoculated in duplicate into Bellco tubes containing Hobson's M2 liquid medium supplemented with flavomycin (0·25–64 µg ml–1). After 48 h incubation at 39 °C, the growth of the cultures (OD650) was compared to that of a control culture, grown in the absence of flavomycin, and the MIC value was determined. The flavomycin sensitivity of the archaeon M. smithii and ruminal fungi was determined in a similar manner, using gas production as an index of activity (Newbold et al., 1988). This method was used because the OD650 of the stationary-phase cultures was too low to be used reliably for the assessment of growth. The flavomycin sensitivity of mixed ruminal protozoa was determined by using the method of Wallace & McPherson (1987).

Bacterial counts.
Most-probable number methods were used for enumeration of total viable anaerobes (Dehority et al., 1989), HAP species (Eschenlauer et al., 2002) and F. necrophorum (Tan et al., 1994). The method was modified to allow quantification to be carried out in microtitre plates. Culture medium (160 µl) was added to sterile microtitre-plate wells in an anaerobic (80 % N2, 10 % CO2, 10 % H2) workstation and 40 µl diluted sample was added in triplicate. Plates were sealed with a sterile adhesive film (Sigma), mixed and incubated for 72 h at 39 °C. The population size of total anaerobes and HAP bacteria was calculated by using most-probable number tables (Alexander, 1982) with values derived from the number of wells that showed positive growth (difference between 0 and 72 h OD650 reading exceeding that of the uninoculated control wells by 0·05). The number of triplicate wells of each dilution that were positive for indole production (tested for with Kovacs reagent; Becton Dickinson) was used to determine the population size of F. necrophorum.

DNA extraction and PCR amplification.
Ruminal digesta samples were washed with 10 ml 20 mM Tris/HCl, 2 mM EDTA (pH 7·0) containing 0·1 g polyvinylpolypyrrolidone prior to the direct extraction of DNA with the chemical and physical cell-lysis method of Tsai & Olson (1991). DNA preparations were treated with RNase A and then purified with Wizard DNA clean-up resin (Promega). PCR amplification across the 16S rRNA V3 region was carried out by using the primers of Muyzer et al. (1993), GC342f (5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3') and 534r (5'-ATTACCGCGGCTGCTGG-3'). Sample DNA (20 ng) was added to a 100 µl reaction mix containing 1 µM each primer, 0·8 mM dNTPs, 1·5 mM MgCl2, 50 mM KCl and 2·5 U Taq DNA polymerase in 10 mM Tris/HCl (pH 9·0). Amplification conditions were 25 cycles with 1 min at 94 °C for denaturation, 1 min at 55 °C for annealing and 2 min at 72 °C for extension, except for 5 min denaturation in the first cycle and 7 min extension in the last cycle. PCR amplification products were visualized on 2 % (w/v) agarose gels prior to DGGE analysis.

DGGE analysis.
DGGE was performed by using the Bio-Rad DCode universal mutation-detection system, following the manufacturer's guidelines. PCR products (20 µl) were loaded onto 8 % (w/v) polyacrylamide gels in 1x TAE [40 mM Tris base, 20 mM acetic acid, 1 mM EDTA (pH 8·3)] that contained a 35–55 % denaturant gradient [100 % denaturant, 7 M urea, 40 % (v/v) deionized formamide]. Electrophoresis was performed at a constant voltage and temperature of 130 V and 60 °C for 5 h. Gels were then stained for 30 min with SYBR gold and the gel image was saved with a Bio-Rad GelDoc 2000 gel-documentation system.

DGGE profiles within the same gels were compared by using similarity trees. Each band position present in the gel was binary-coded for its presence or absence within a lane and each lane was compared by using a similarity matrix. Trees were constructed by using the Hamming distance values generated for each comparison (indicating the number of bands that differed between lanes) as an input for the NEIGHBOR program (PHYLIP version 3.6; Felsenstein, 2002).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Influence of flavomycin on ruminal fermentation and metabolism
Flavomycin supplementation of the diet caused significant decreases in the ruminal concentrations of ammonia and total VFA (Table 3). No significant interaction between flavomycin and the sampling time after feeding occurred (data not shown). The concentrations of most individual acids decreased significantly (acetic, butyric, propionic, valeric and isobutyric), apart from isovaleric and caproic acids. No change in the acetate : propionate ratio was observed. Ruminal lactate concentrations increased significantly as a result of supplementation, but only by a small amount (0·46 mM). No significant changes occurred with respect to the pH of the ruminal digesta (control 6·40, flavomycin 6·32; P=0·15). The fermentation parameters did not differ significantly between 7, 8, 14 and 15 days of flavomycin supplementation.


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Table 3. Influence of dietary flavomycin on ruminal fermentation products

 
Ammonia-production rates were measured in rumen digesta samples removed from animals receiving the control diet in order to determine the effects of flavomycin on deamination of endogenous substrates and of PCH by a batch-culture method (Table 4). Ammonia-production rates in the different sheep were highly variable. As a consequence, although both rates of ammonia production decreased with flavomycin, differences were not statistically significant. The effects on rates were comparable to those obtained with monensin (Table 4).


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Table 4. Influence of flavomycin and monensin on rates of ammonia production in ruminal digesta in vitro

 
Sensitivity of ruminal micro-organisms to flavomycin in vitro
The growth-inhibitory effect of flavomycin on pure cultures of rumen bacteria was highly selective (Table 1). Several species of HAP (‘hyper-ammonia producing’ or ‘ammonia-hyper-producing’) (Russell et al., 1991; Attwood et al., 1998) bacteria were sensitive to the antibiotic (F. necrophorum, a Desulfomonas-like species and Peptostreptococcus anaerobius), as was ‘Atopobium oviles’ and all of the Fibrobacter species tested. Gas production during growth of ruminal fungi and of the archaeon M. smithii were not affected by flavomycin, nor was the bacteriolytic activity of mixed ruminal protozoa (data not shown).

In vitro susceptibility tests of F. necrophorum strains isolated from liver abscesses demonstrated that these were also sensitive to flavomycin (Table 2). Type strains of other Fusobacterium species isolated from the gut were also tested. Fusobacterium necrogenes, Fusobacterium pseudonecrophorum and Fusobacterium perfoetens were susceptible to flavomycin; however, the latter two required much higher concentrations of flavomycin to inhibit growth strongly. F. pseudonecrophorum and Fusobacterium varium have been suggested to be synonymous (Bailey & Love, 1993); however, their sensitivity to flavomycin differed. The rates of ammonia production varied in different Fusobacterium strains and also differed according to the growth medium (Table 2). The richer Hobson's M2 liquid medium gave a higher specific activity of ammonia formation than a simpler medium. The rates of ammonia production were comparable to those of Clostridium aminophilum, a well-known HAP bacterium (Table 2).

Effect of flavomycin on ruminal viable bacterial counts
Sheep on the control diet and receiving flavomycin were killed and the total anaerobic viable count and numbers of F. necrophorum in ruminal digesta and associated with ruminal wall tissue were determined (Table 5). F. necrophorum as a proportion of the total population was an order of magnitude greater in wall tissue than in digesta (0·27x10–4 in digesta, 3·0x10–4 in wall-tissue samples). Flavomycin had no significant influence on numbers of F. necrophorum in either sample type; however, the variation in numbers was very high. In order to investigate the influence of flavomycin on viable counts of both F. necrophorum and HAP bacteria, counts were made before and after the introduction of flavomycin and the results were expressed as a proportion of the total viable counts (Fig. 1). The proportion of both categories of bacteria decreased following the introduction of flavomycin. HAP populations had the biggest day-to-day variations, whereas variation in numbers of F. necrophorum was mainly between animals.


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Table 5. Effect of dietary flavomycin on most-probable numbers of anaerobic bacteria and of Fusobacterium species in ruminal digesta and wall tissue of sheep receiving a grass hay/concentrate diet

 


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Fig. 1. Influence of dietary flavomycin on bacterial numbers in ruminal digesta from sheep. The number of viable F. necrophorum (a) and HAP bacteria (b) in the ruminal digesta of four different sheep (A–D) was calculated as a proportion of the total viable count in each animal, measured before (black bars) and after 2 weeks (grey bars) of flavomycin supplementation. Error bars represent SD of values generated from rumen digesta samples on two consecutive days during the control (–1 and 0 days) and flavomycin-treatment (14 and 15 days) periods.

 
Effect of flavomycin on the ruminal bacterial community as assessed by DGGE
16S rRNA gene analysis of the ruminal bacterial flora using DGGE confirmed that flavomycin caused distinct changes in the bacterial population (Fig. 2). However, differences in microbiota composition between animals still remained larger than any treatment effect. After 7 days of flavomycin supplementation, the population structure did not fluctuate greatly, as was also indicated by the stabilization of the ruminal fermentation parameters.



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Fig. 2. DGGE profiles of the ruminal bacterial population in digesta taken from flavomycin-supplemented sheep. Ruminal digesta samples from two cannulated adult sheep from before (–1 and 0 days) and after (7, 8, 14 and 15 days) flavomycin dietary supplementation were analysed. DNA was extracted from rumen digesta samples and the 16S rRNA gene was amplified by PCR and separated by DGGE. DNA was visualized by using SYBR gold nucleic acid gel stain. DGGE profiles of samples from different sheep were compared by using similarity trees. Each band position present in the gel was binary-coded for its presence or absence within a lane and each lane was compared by using a similarity matrix. Trees were constructed by using the Hamming distance values generated for each comparison (indicating the number of bands that differed between lanes) as an input for the NEIGHBOR program [PHYLIP version 3.6 (Felsenstein, 2002)].

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although flavomycin has been used in livestock production for many years, its mode of action in growth promotion has not been clearly established. As with the majority of studies in ruminants in vivo (Rowe et al., 1982; Galbraith et al., 1983; Febel et al., 1988; Alert et al., 1991), it was found here that flavomycin did not alter the proportion of different VFA, although a decrease in their concentration did occur. The decrease in the total VFA concentration correlated with a non-significant decrease in total viable anaerobic bacteria. Whether this change in number can solely account for the large change in acetate concentration is not clear. Several of the bacteria found to be flavomycin-sensitive in vitro do produce acetate, including ‘A. oviles’, Fibrobacter succinogenes and P. anaerobius. The numbers of these species in the rumen may be large enough to account for at least part of the decrease in acetate, if their numbers were decreased by flavomycin in vivo. However, as a large majority of rumen bacteria remains to be cultivated (Edwards et al., 2004), trying to explain in vivo effects only on the basis of known cultivated organisms will always be a compromise until more is known regarding their metabolic activities.

The lack of response to flavomycin in the proportion of different VFA contrasts with the effects of ionophores, including monensin, salinomycin, narasin and tetronasin, which promote increased propionate formation (Nagaraja et al., 1997). Thus, a different mode of action in terms of antimicrobial activity might be expected for flavomycin in comparison with the ionophores. Additionally, decreased tissue-turnover rates have been observed in sheep receiving flavomycin (MacRae et al., 1999; Edwards et al., 2005); as flavomycin is unlikely to have a direct effect on the animal itself, based on in vivo studies with germ-free animals (Reidel et al., 1974), this suggested that one mechanism of action may involve bacteria–tissue interactions.

The spectrum of antimicrobial activity found with flavomycin did indeed differ markedly from the ionophores. Whereas ionophores inhibit Gram-positive bacteria in general (Nagaraja et al., 1997), only a relatively narrow group of organisms was affected at flavomycin concentrations of <2 µg ml–1, which is considered to be the theoretical maximal concentration of flavomycin in the rumen (Van Nevel & Demeyer, 1987).

The Gram-negative, cellulolytic Fibrobacter species were among the most sensitive to flavomycin in vitro. Inhibition of cellulolysis would be detrimental to production efficiency. However, flavomycin does not appear to suppress cellulose and fibre degradation in vitro or in vivo (Cafantaris, 1981; Rowe et al., 1982; Bedo et al., 1984), so it is probable that the cellulolytic activities of Ruminococcus species, ruminal fungi and ciliate protozoa may compensate for a decrease in fibrobacter numbers.

The main mechanism of action of flavomycin seems likely to involve two groups of bacteria, namely HAP species and the Gram-negative fusobacteria. Indeed, the results of the present study and also those of Attwood et al. (1998) indicate that Fusobacterium species have a very high rate of deamination and should also be considered HAP species. Some HAP bacteria were sensitive to flavomycin at concentrations of <2 µg ml–1, including a Desulfomonas-like species (Eschenlauer et al., 2002), P. anaerobius (Russell et al., 1988) and F. necrophorum (Attwood et al., 1998). A. oviles’ was also highly sensitive to flavomycin; however, the deamination capacity of this acetate-producing bacterium is low, despite the fact that it was isolated under the same conditions as the Desulfomonas-like species (Eschenlauer et al., 2002). These bacteria, which have high specific activities of deamination, cause the excessive conversion of dietary amino acids to ammonia (Russell et al., 1991). Here, lower ammonia concentrations were observed in vivo with flavomycin, and ammonia-production rates tended to be lower in digesta with added flavomycin. Van Nevel & Demeyer (1987) reported that flavomycin caused a decrease in ammonia production from casein in vitro, without affecting the degradation of casein itself. The ability of flavomycin to restrict the conversion of amino acids to ammonia, through direct suppression of some ruminal HAP bacteria, would be consistent with these findings. Decreases in HAP bacteria as a proportion of the total bacterial population were also observed here, consistent with this proposed mechanism.

F. necrophorum was much more abundant on the ruminal wall than in ruminal digesta and, although no decrease in the ruminal wall population could be demonstrated in response to flavomycin, the numbers in digesta appeared to fall in response to the introduction of flavomycin into the diet. The ruminal lactate concentration increased as a result of flavomycin treatment. Although the concentrations were small, this may be consistent with F. necrophorum being a lactate utilizer.

F. necrophorum is generally regarded as an opportunistic pathogen (Langworth, 1977). It attaches to ruminal epithelial cells (Kanoe & Iwaki, 1987; Takayama et al., 2000), where it can proliferate and cause inflammation and necrotic lesions. Thus, suppression of F. necrophorum is also consistent with a lower rate of tissue turnover (Edwards et al., 2005). As Fusobacterium species are present throughout the digestive tract in sheep (Edwards, 2003) and can also be found in pigs and poultry (Langworth, 1977), the effectiveness of flavomycin elsewhere in the digestive tract of ruminants and in other species may result from the suppression of tissue invasion by Fusobacterium species. It is well-established that the problems of liver abscesses in cattle result from invasion by F. necrophorum (Nagaraja & Chengappa, 1998).

Both the suppression of HAP activity and gut-tissue turnover may explain why flavomycin has been reported to have favourable effects on nitrogen metabolism in ruminants (Bedo et al., 1984; Alert et al., 1991). Flavomycin enhances the absorption of amino acids in the gastrointestinal tract of fattening cattle, based on total amino acid balance data (Alert et al., 1991). Paired with the decreased protein turnover observed in the gut, decreasing the tissue requirements for amino acids, flavomycin would appear to have the ability to increase the amount of amino acids available to the animal (MacRae et al., 1999; Wallace et al., 2001). This mechanism also offers a potential explanation for the ability of flavomycin to increase wool growth and the protein content of milk (Hamann, 1983; Murray et al., 1990, 1992).

The effects of flavomycin on the bacterial community were also investigated by molecular-community analysis using DGGE. Analysis demonstrated how different bacterial-community structures can be between individual animals and that this difference was maintained, despite the treatment of animals with flavomycin. At present, we have no real clue as to the animal factors that regulate the bacterial community so individually. Such factors may be important to find in order to understand how the host animal determines its own gut microflora.

In conclusion, this study demonstrated that flavomycin has the ability to alleviate metabolic burdens that are imposed by the flora of the rumen, through the direct suppression of ruminal HAP bacteria and F. necrophorum. As Fusobacterium species from pig and poultry were also sensitive to flavomycin, alleviation of immune challenges and ammonia-associated toxicity may also play a role in the promotion of growth in non-ruminant animals (Tan et al., 1996; Anderson et al., 2000).


   ACKNOWLEDGEMENTS
 
The RRI receives funding from the Scottish Executive Environmental and Rural Affairs Department. J. E. E. acknowledges the award of a studentship from the Aberdeen Research Consortium. B. Bequette and L. Bruce helped in the provision of tissue and digesta samples. The gift of bacterial cultures by T. G. Nagaraja and G. T. Attwood is gratefully acknowledged.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ahrens, F. (1987). The effect of flavomycin on rumen physiological and caecal parameters and on milk output and milk constituents in cows: final report. IS Research Society for Experimental Animal Physiology and Animal Nutrition.

Alert, H. J., Meier, H. & Poppe, S. (1991). Studies on digestion physiology after use of flavomycin in the fattening of young beef bulls: trial report. Trial report, University of Rostock, Germany.

Alexander, M. (1982). Most probable number method for microbial populations. In Methods of Soil Analysis, Part 2, pp. 815–820. Agronomy Monograph no. 9, 2nd edn. Madison, WI: American Society of Agronomy.

Anderson, D. B., McCracken, V. J., Aminov, R. I., Simpson, J. M., Mackie, R. I., Verstegen, M. W. A. & Gaskins, H. R. (2000). Gut microbiology and growth-promoting antibiotics in swine. Nutr Abstr Rev 70, 101–108.

Attwood, G. T., Klieve, A. V., Ouwerkerk, D. & Patel, B. K. C. (1998). Ammonia-hyperproducing bacteria from New Zealand ruminants. Appl Environ Microbiol 64, 1796–1804.[Abstract/Free Full Text]

Bailey, G. D. & Love, D. N. (1993). Fusobacterium pseudonecrophorum is a synonym for Fusobacterium varium. Int J Syst Bacteriol 43, 819–821.

Bauer, F. & Dost, G. (1971). Flavomycin, the first antibiotic intended exclusively as a food supplement. In The Blue Book for the Veterinary Profession, vol. 21, pp. 87–92.

Bedo, S., Bodis, L., Ravasz, T. & Kovacs, G. (1984). Effect of growth promoters on the metabolism of young lambs. Allattenyesztes Takarmanyozas 33, 139–148.

Cafantaris, B. (1981). The effect of antibiotic supplements on microbial fermentation in the ruminal fluid in vitro. PhD thesis, University of Hohenheim, Germany.

Dehority, B. A., Tirabasso, P. A. & Grifo, A. P., Jr (1989). Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium. Appl Environ Microbiol 55, 2789–2792.[Medline]

Edwards, J. E. (2003). Characterisation of the effect of flavomycin on the rumen microflora. PhD thesis, University of Aberdeen, UK.

Edwards, J. E., Wallace, R. J. & McEwan, N. R. (2002). The growth promoting mode of action of flavomycin in ruminants. Reprod Nutr Dev 42 (Suppl. 1), S55.[CrossRef]

Edwards, J. E., McEwan, N. R., Travis, A. J. & Wallace, R. J. (2004). 16S rDNA library-based analysis of ruminal bacterial diversity. Antonie Van Leeuwenhoek 86, 263–281.[CrossRef][Medline]

Edwards, J. E., Bequette, B. J., McKain, N., McEwan, N. R. & Wallace, R. J. (2005). Influence of flavomycin on microbial numbers, microbial metabolism and gut tissue protein turnover in the digestive tract of sheep. Br J Nutr (in press).

Eschenlauer, S. C. P., McKain, N., Walker, N. D., McEwan, N. R., Newbold, C. J. & Wallace, R. J. (2002). Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. Appl Environ Microbiol 68, 4925–4931.[Abstract/Free Full Text]

Febel, H., Szelenyi, M., Jecsai, J. & Juhasz, B. (1988). Effect of salinomycin, flavomycin and avoparcin on some physiological traits of growing lambs, with particular respect to rumen fermentation. Acta Vet Hung 36, 69–80.[Medline]

Felsenstein, F. (2002). PHYLIP (Phylogeny Inference Package) version 3.6a. Seattle: University of Washington.

Galbraith, H., Scaife, J. R. & Lowe, R. H. (1983). Response of growing bulls to the food additives, salinomycin and flavomycin. Anim Prod 36, 527–528.

Hamann, J. (1983). Flavomycin treatment in dairy cows - results from a field study. Tierarztl Umsch 38, 90–98.

Hobson, P. N. (1969). Rumen bacteria. In Methods in Microbiology, vol. 3B, pp. 133–149. Edited by J. R. Norris & D. W. Ribbons. London: Academic Press.

Hochella, N. J. & Weinhouse, S. (1965). Automated lactic acid determination in serum and tissue extracts. Anal Biochem 10, 304–317.[CrossRef][Medline]

Huber, G. (1979). Moenomycin and related phosphorus-containing antibiotics. In Mechanisms of Action of Antibacterial Agents (Antibiotics vol. 5 part 1), pp. 135–153. Edited by F. E. Hahn. New York: Springer.

Kanoe, M. & Iwaki, K. (1987). Adherence of Fusobacterium necrophorum to bovine ruminal cells. J Med Microbiol 23, 69–73.[Abstract]

Langworth, B. F. (1977). Fusobacterium necrophorum: its characteristics and role as an animal pathogen. Bacteriol Rev 41, 373–390.[Medline]

MacRae, J. C., Bruce, L. A. & Yu, F. (1999). The effect of Flavomycin on gastrointestinal leucine metabolism and liveweight gain in lambs. S Afr J Anim Sci 29, 243–244.

Masoero, F., Prandini, A. & Fiorentini, L. (1991). Flavomycin/physiology trial in dairy cattle in vitro. Report of first trial phase in vitro with RUSITEC. Catholic University of Sacro Cuore, Italy.

McKain, N., Edwards, J. E., Wallace, R. J., Edwards, S., Bruce, L., Bequette, B. J. & MacRae, J. C. (2000). Effects of flavomycin in the gastrointestinal tract of sheep. Reprod Nutr Dev 40, 222.

Murray, P. J., Rowe, J. B. & Aitchison, E. M. (1990). The influence of protein quality on the effect of flavomycin on wool growth, liveweight change and rumen fermentation in sheep. Austr J Agric Res 41, 987–993.

Murray, P. J., Winslow, S. G. & Rowe, J. B. (1992). Conditions under which flavomycin increases wool growth and liveweight gain in sheep. Austr J Agric Res 43, 367–377.[CrossRef]

Muyzer, G., de Waal, E. C. & Uitterlinden, A. G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59, 695–700.[Abstract]

Nagaraja, T. G. & Chengappa, M. M. (1998). Liver abscesses in feedlot cattle: a review. J Anim Sci 76, 287–298.[Abstract/Free Full Text]

Nagaraja, T. G., Newbold, C. J., Van Nevel, C. J. & Demeyer, D. I. (1997). Manipulation of ruminal fermentation. In The Rumen Microbial Ecosystem, 2nd edn, pp. 523–632. Edited by P. N. Hobson. New York: Blackie.

Newbold, C. J., Wallace, R. J., Watt, N. D. & Richardson, A. J. (1988). Effect of the novel ionophore tetronasin (ICI 139603) on ruminal microorganisms. Appl Environ Microbiol 54, 544–547.[Medline]

Newbold, C. J., Wallace, R. J., Chen, X. B. & McIntosh, F. M. (1995). Different strains of Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in vitro and in sheep. J Anim Sci 73, 1811–1818.[Abstract/Free Full Text]

Rebolini, O., Gallazzi, D. & Valerani, L. (1982). Use of flavophospholipol as a growth promotant in feeds for fattening rabbits. Coniglicoltura 19, 57–60.

Reidel, G., Reiter, H. & Losch, U. (1974). Effect of oral flavophospholipol (FPL) on the growth of germfree chickens. Z Tierphysiol 32, 328–334.

Rowe, J. B., Morrell, J. S. & Broome, A. W. J. (1982). Flavomycin as a ruminant growth promoter – investigation of the mode of action. Proc Nutr Soc 41, A56.

Russell, J. B., Strobel, H. J. & Chen, G. (1988). Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production. Appl Environ Microbiol 54, 872–877.[Medline]

Russell, J. B., Onodera, R. & Hino, T. (1991). Ruminal protein fermentation: new perspectives on previous contradictions. In Physiological Aspects of Digestion and Metabolism in Ruminants, pp. 681–697. Edited by T. Tsuda, Y. Sasaki & R. Kawashima. San Diego: Academic Press.

Takayama, Y., Kanoe, M., Maeda, K., Okada, Y. & Kai, K. (2000). Adherence of Fusobacterium necrophorum subsp. necrophorum to ruminal cells derived from bovine rumenitis. Lett Appl Microbiol 30, 308–311.[CrossRef][Medline]

Tan, Z. L., Nagaraja, T. G. & Chengappa, M. M. (1994). Selective enumeration of Fusobacterium necrophorum from the bovine rumen. Appl Environ Microbiol 60, 1387–1389.[Abstract]

Tan, Z. L., Nagaraja, T. G. & Chengappa, M. M. (1996). Fusobacterium necrophorum infections: virulence factors, pathogenic mechanism and control measures. Vet Res Commun 20, 113–140.[Medline]

Tsai Y.-L. & Olson, B. H. (1991). Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57, 1070–1074.[Medline]

van Heijenoort, J. (2001). Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology 11, 25R–36R.[Abstract/Free Full Text]

Van Heijenoort, Y., Derrien, M. & Van Heijenoort, J. (1978). Polymerization by transglycosylation in the biosynthesis of the peptidoglycan of Escherichia coli K 12 and its inhibition by antibiotics. FEBS Lett 89, 141–143.[CrossRef][Medline]

Van Nevel, C. V. & Demeyer, D. (1987). Modification of rumen protein fermentation in vitro by antibiotics. Med Fac Landbouww Rijksuniv Gent 52, 1691–1701.

Wallace, R. J. (1978). Control of lactate production by Selenomonas ruminantium: homotropic activation of lactate dehydrogenase by pyruvate. J Gen Microbiol 107, 45–52.[Medline]

Wallace, R. J. & McPherson, C. A. (1987). Factors affecting the rate of breakdown of bacterial protein in rumen fluid. Br J Nutr 58, 313–323.[Medline]

Wallace, R. J., Newbold, C. J., Bequette, B. J., MacRae, J. C. & Lobley, G. E. (2001). Increasing the flow of protein from ruminal fermentation - review. Asian-Australas J Anim Sci 14, 885–893.

Wallhausser, K. H., Nesemann, G., Prave, P. & Steigler, A. (1965). Moenomycin, a new antibiotic. I. Fermentation and isolation. Antimicrob Agents Chemother 5, 734–736.

Whitehead, R., Cooke, G. H. & Chapman, B. T. (1967). Problems associated with the continuous monitoring of ammoniacal nitrogen in river water. Automat Anal Chem 2, 377–380.

Received 3 September 2004; revised 16 November 2004; accepted 22 November 2004.



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