Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Correspondence
R. John Wallace
rjw{at}rri.sari.ac.uk
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ABSTRACT |
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Present address: Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK.
Present address: AgResearch, Private Bag, Palmerston North, New Zealand.
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INTRODUCTION |
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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
).
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METHODS |
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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 day1. 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 (D1D10) 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 ml1 in ethanol (giving a final flavomycin concentration of 2 µg ml1) or 16 µl monensin (10 mg ml1) 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 l1.
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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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 3555 % 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).
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RESULTS |
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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 (0·27x104 in digesta, 3·0x104 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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DISCUSSION |
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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 bacteriatissue 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 ml1, 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 ml1, 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
).
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ACKNOWLEDGEMENTS |
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Received 3 September 2004;
revised 16 November 2004;
accepted 22 November 2004.
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