Effect of hen age, Bio-Mos and Flavomycin on susceptibility of turkey poults to oral Escherichia coli challenge
Published: November 2, 2007
By: A.S. FAIRCHILD, J.L. GRIMES, F.W. EDENS, M.J. WINELAND, F.T. JONES, and A.E. SEFTON (Courtesy of Alltech Inc.)
At week 1 and week 3, one bird per pen (n=128) was chosen for bacterial sampling. Aliquots from liver and intestinal tissue were spiral plated on the following media: Eosin Methylene Blue agar (EMB) for total coliforms, Tryptic Soy agar (TSA) for total aerobic bacteria, and lactobacillus selection agar (LBS) for Lactobacillus sp. Any sample yielding isolated E. coli colonies was subcultured and sent to the E. coli Reference Center, University Park, PA, for O serotyping.
Individual body weight and feed consumption by pen were recorded on a weekly basis. Poult mortality was recorded on a daily basis. Feed conversion and body weight gains were calculated. Results indicate interactions of hen age with Bio-Mos and Flavomycin for bacterial counts for intestinal and liver tissues for both week 1 and week 3 (P£0.05). Under E. coli challenge, Bio-Mos and Flavomycin (P<0.05) improved poult growth during week 2 while Flavomycin improved poult growth through week 3 (P£0.05). Cumulative three week body weight gains for unchallenged poults were improved by both Bio-Mos and Flavomycin (P£0.05). It may be concluded that dietary Bio-Mos and Flavomycin can improve the overall performance of poults especially when they are faced with an E. coli challenge during the first few weeks of life.
INTRODUCTION
Antimicrobials have traditionally been used in the poultry industry to improve health and performance of birds by decreasing or altering the bacterial populations present in the digestive tract. This has been done during the grow-out period to protect the animal from pathogenic organisms and to increase weight gains and improve meat quality.
Antibiotics have come under increasing scrutiny by researchers and consumers alike because there are increasing percentages of antibioticresistant bacteria (including pathogenic strains) seen after the inclusion of antibiotics in the feed. Antibiotic resistance displayed by field E. coli isolates from North Carolina commercial turkey farms has been reported (Fairchild et al., 1998), including resistance to enrofloxacin, one of the most recent antibiotics approved for use in poultry (Figures 1 and 2).
Most of the antibiotics used as growth promoters have no significant approved claims to control disease (Gustafson and Bowen, 1997). Debate over resistance seen among Gram-negative bacteria such as E. coli and salmonella has generated the strongest objection to antibiotic use (Evagelisti et al., 1975; Scioli et al., 1983; Gustafson and Bowen, 1997). It has been reported that antibiotic resistance to indigenous E. coli in poultry has remained at a relatively high level since the 1950s (Gustafson and Bowen, 1997). Furthermore, claims have been made that the use of these antibiotics could disrupt normal gut microflora (Surawicz et al., 1989).
Consequently, other approaches, such as competitive exclusion (CE) treatments, have been developed to counter the growth-depressing effects that certain strains of bacteria elicit in poultry. One type of CE aims at the development of a protective barrier bacteria population in the digestive tract that prevents the colonization of unfavorable (growth depressing and/or pathogenic) microorganisms.
Some cultures have included Lactobacillus sp. (Francis et al., 1978) or undefined normal avian gut flora (Nurmi and Rantala, 1973). Another CE type exploits the presence of mannose-specific fimbriae on unfavorable Gram negative bacteria such as strains of E. coli and salmonella. These bacteria use the fimbriae to attach to and then colonize the intestinal wall. Mannanoligosaccharides (MOS), derived from mannans on yeast cell surfaces, act as high affinity ligands (decoys), offering a competitive binding site for the bacteria (Ofek et al., 1977). Pathogens with the mannose-specific fimbriae adsorb to the MOS (decoy) instead of attaching to intestinal epithelial cells, and therefore move through the intestine without colonization. Newman (1994) reported that the presence of dietary MOS in the intestinal tract removed pathogenic bacteria that could attach to the lumen of the intestine in this manner.
This might provide a more favorable environment for nutrient utilization by the bird (Savage and Zakrzewska, 1996). The present study was designed to determine whether aMOSproduct, Bio-Mos, and an antibiotic used for growth enhancement, Flavomycin, alone or in combination would alter the numbers of intestinal tract and liver bacteria or improve performance characteristics of poults from hens of two different ages challenged with E. coli from 1-21 days of age.
MATERIALS AND METHODS
PREPARATION OF ORAL E. COLI CHALLENGE CULTURES
Five E. coli cultures (including one pathogenic culture), isolated from North Carolina commercial turkey farms, were chosen based on strong resistance to 8 different antibiotics available to the poultry industry and strong agglutination with Bio-Mos (Figures 1 and 2). A growth curve was performed to determine the point at which the cultures reached 108 CFU/ml, which was the gavage dosage used in this study. Each was then grown independently overnight and mixed before administration. Serotypes used included O:2, O:19, O:88, and O:159. Additional cultures of the bacteria were grown throughout the study in order to provide supplemental oral challenge via water troughs on days 4, 8, 12, 16, and 20.
EXPERIMENTAL BIRDS AND CHALLENGE PROTOCOL
Day-of-hatch male poults (British United Turkeys, Lewisburg, WV) (n=896) from both a young (33 weeks of age, WOA) and old (58 WOA) breeder flock were placed in four rooms with each room containing two batteries (Petersime Incubator Co., Gettysburg, OH ) with wire mesh floors. Sixteen pens were used in each battery with seven birds placed in each pen. At placement, poults were gavaged orally with either 1 ml of E. coli suspended in Veal Infusion broth (VI, Difco Laboratories, Detroit, MI) at a concentration of 108 CFU/ml, or 1 ml of sterile VI broth.
On days in which additional E. coli challenge was supplied, 0.1 ml of E. coli suspension was added to the water troughs (2.0 liters) so that the final concentration of E. coli in the troughs was 106 CFU/ml.Water troughs were rinsed once per week and not disinfected to facilitate any bacterial growth. Two rooms each were used to house the challenged and unchallenged poults providing for a split plot arrangement of treatments. Control birds (without E. coli challenge) were gavaged first to lessen the likelihood of cross contamination.
EXPERIMENTAL BIRDS AND CHALLENGE PROTOCOL
Day-of-hatch male poults (British United Turkeys, Lewisburg, WV) (n=896) from both a young (33 weeks of age, WOA) and old (58 WOA) breeder flock were placed in four rooms with each room containing two batteries (Petersime Incubator Co., Gettysburg, OH ) with wire mesh floors. Sixteen pens were used in each battery with seven birds placed in each pen. At placement, poults were gavaged orally with either 1 ml of E. coli suspended in Veal Infusion broth (VI, Difco Laboratories, Detroit, MI) at a concentration of 108 CFU/ml, or 1 ml of sterile VI broth.
On days in which additional E. coli challenge was supplied, 0.1 ml of E. coli suspension was added to the water troughs (2.0 liters) so that the final concentration of E. coli in the troughs was 106 CFU/ml.Water troughs were rinsed once per week and not disinfected to facilitate any bacterial growth. Two rooms each were used to house the challenged and unchallenged poults providing for a split plot arrangement of treatments. Control birds (without E. coli challenge) were gavaged first to lessen the likelihood of cross contamination.
DIETARY TREATMENTS
The poults were fed a turkey starter ration (Table 1). The same ration was used for four dietary treatments: control, Bio-Mos (2 lb/ton feed, Alltech, Inc.), Flavomycin (2 g active ingredient/ton feed, Hoechst Roussel Vet) and Bio-Mos plus Flavomycin. De-ionized water and feed were provided ad libitum.
POULT SUSCEPTIBILITY TO E.COLI CHALLENGE
Figure 1. North Carolina field Escherichia coli isolate showing moderate and full resistance to eight antibiotics; none were categorized as susceptible using test guidelines. (A: Sulfadimethoxine 23.8 mcg /Ormetoprim 1.2 mcg; B: Chlortetracycline 30 mcg; C: Clindamycin 2 mcg; D: Gentamycin 10 mcg; E: Penicillin 10 units; F: Bacitracin 10 units; G: Neomycin 30 mcg; H: Enrofloxacin 5 mcg. All strengths listed as amount in each disk.)
Figure 2.Second North Carolina field Escherichia coli isolate showing a more susceptible profile. A: Sulfadimethoxine/Ormetoprim; B: Chlortetracycline; C: Clindamycin; D: Gentamycin; E: Penicillin; F: Bacitracin; H: Enrofloxacin. Isolate is resistant to B, C, D, and F; susceptible to A, E, and H. All strengths are same for those used for Figure 1. 1
SAMPLING, ENUMERATION, AND PERFORMANCE DATA
At weeks 1 and 3, one bird per pen (128 birds/week) was chosen at random for microbial analyses. The selected poults were weighed, euthanized, and liver and intestinal tract posterior to the duodenal loop were aseptically removed. The samples were then brought to a 1:10 dilution by weight in 0.85% saline and mechanically massaged (Stomacher® Model 80 Mark II Laboratory Blender, Tekmar-Dohrmann, Cincinnati, OH) for 1minute.
All samples were finally diluted to appropriate levels for plating. An aliquot from each sample (100 μl) was spiral plated (Model D, Spiral Systems Inc., Cincinnati, OH) on the following: Eosin Methylene Blue agar (EMB) for the isolation and enumeration of total coliforms, lactobacillus selection agar (LBS) for the enumeration of Lactobacillus sp. and Tryptic Soy agar (TSA) for the enumeration of total aerobic bacteria. Any EMB plate yielding isolated Escherichia coli colonies was subcultured and sent to the E. coli Reference Center, University Park, PA, for O serotyping to see if any serotypes retrieved matched those administered.
Individual body weights and feed consumption by pen were measured on a weekly basis. Weekly and cumulative body weight gains, feed conversion ratios, and feed:gain ratios were calculated.
EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS
Within each gavage treatment group, birds were subjected to a randomized complete block arrangement of 2 hen ages x 2 levels of Bio-Mos x 2 levels of Flavomycin. All feed treatments were balanced on each side of each battery. Bacterial counts were transformed to their Log10 values and mortality percentages were subjected to arc sine transformation before statistical analysis. All data were analyzed using the General Linear Models Procedures of SAS.
The effects of E. coli challenge, hen age, Bio-Mos and Flavomycin were regressed on bacterial enumeration and poult performance. Treatment means were separated using the Least Significant Difference procedure of SAS. Any discussion of differences between treatment means in the following section are to be considered significant (P£ 0.05) unless stated otherwise.
RESULTS AND DISCUSSION
PERFORMANCE
Greater body weight gains weremade during weeks one and two for poults challenged with E. coli when the poults were fed a diet supplemented with Bio-Mos or Flavomycin (Table 2). During week one, a diet supplemented with both Bio-Mos and Flavomycin in combination resulted in lower poult growth than for the control diet or diets with a single supplement.
Poults fed the combination diet were intermediate in growth during week two and had equivalent growth during week three.Weekly body weight gains of poults without E. coli challenge were improved by both Bio-Mos and Flavomycin at the end of week two and by Flavomycin at the end of week three (Table 2). During week one, poults from old hens had significantly greater body weight gains than poults from young hens (70.5 vs. 58.9 g) when not challenged with E. coli. Under E. coli challenge during week one and for both challenge groups for weeks two and three, poults from old and young hens gained the same.
Cumulative body weight gains (Table 3) following E. coli challenge were not significantly different at weeks two or three but demonstrated a pattern similar to that observed for weekly gains (Table 2). Cumulative body weight gains of poults not challenged with E. coli were improved when the diet was supplemented with Bio-Mos or Flavomycin (Table 3).
Weekly body weights of poults with and without E. coli challenge are summarized in Table 4. Following E. coli challenge, all poults had significantly higher body weights at day 7 when either Bio-Mos or Flavomycin was added to the diet, but not when the diet contained both additives.
At day 14, poults fed Bio-Mos were heavier than poults fed no additive or both additives; while poults fed Flavomycin were intermediate in weight between Bio-Mos and control poults. At day 21, there were no significant differences in body weights of poults following E. coli challenge; although the numerical improvements for body weights in the presence of either feed treatment was greater than that observed at 14 days.
For those poults not receiving an E. coli challenge, poults fed diets with Flavomycin or Bio-Mos plus Flavomycin were heavier at days 14 and 21, while those fed Bio-Mos were intermediate at day 14 but not different from controls at day 21 (Table 4). Poults from old hens weighed more than poults from young hens at day 7 for the control (125.1 vs. 112.7 g) and Bio-Mos (126.9 vs. 116.8 g) diets (Table 4). Poults from old hens weighed more than poults from young hens over all diets at day 7 (125.4 vs. 116.5 g) and day 14 (316.1 vs. 306.9 g). However, by day 21, there were no differences in body weights between poults from young and old hens (Table 4).
There were no differences in feed conversion between dietary or hen age treatments. Mean three week feed conversion for E. coli challenged poults was 1.42 (±0.03), and for unchallenged poults, 1.33 (±0.03). For poults receiving E. coli challenge, there was a significantly higher cumulative mortality (Table 5) in young poults fed the diet containing Bio-Mos and Flavomycin in combination, with mortality for the remaining treatments being intermediate between the control diet and the diet with both supplements. No other significant differences were observed.
The results of the performance data support previous findings in which feeding Bio-Mos or Flavomycin significantly improved body weight and weight gains of turkeys (Savage et al., 1996; Savage et al., 1997; Buresh et al., 1986; Caston and Leeson, 1992), and extend those findings to improved body weights and weight gains of poults fed Bio-Mos with additional stress (Sims and Sefton, 1999). Sims and Sefton (1999) reared tom turkeys to 18 weeks of age on used turkey litter and fed diets with Bacitracin Methylene Disalycylate (BMD) and Bio-Mos alone and in combination along with a control diet. There were no significant differences in body weights at 6 or 12 weeks of age (Table 6).
However, at both 15 and 18 weeks of age, turkeys fed BMD plus Bio-Mos were heavier (P£0.05) than control fed birds while the birds fed either of the feed additives alone were intermediate in body weight (Table 6). Also, at 18 weeks of age, birds fed Bio-Mos or BMD alone were heavier than control birds but were not as heavy as those fed Bio-Mos and BMD in combination (P£0.05).
In addition, feed conversion was improved (P£0.05) at 18 weeks of age for those birds fed Bio-Mos plus BMD compared to control fed birds, while those fed either Bio-Mos or BMD alone were intermediate in feed conversion (Table 7). Even though the effects occurred at different age periods, the current study and that reported by Sims and Sefton (1999) agree in that poults challenged with a disease agent benefit when Bio-Mos is added to the diet and this benefit is comparable to antibiotic growth promotants such as Flavomycin or BMD.
However, this study did not show significant improvements in feed efficiencies in poults not challenged with E. coli for any treatment diet, which agrees with previously reported studies (Savage et al., 1996; Savage et al., 1997; Buresh et al., 1986; Caston and Leeson, 1992).
Sims (1998) and Sims et al. (1998) reported similar findings with broiler chicks. Broiler chicks were reared in floor pens to 49 days of age using typical broiler diets with either Bio-Mos or BMD added to the diet and compared to a control diet. Bio-Mos was added at a rate of 2 lb/ton in the starter and 1 lb/ton in the grower and finisher. BMD was added at a rate of 50 g/ton to the starter and 25 g/ton in the grower and finisher. All diets contained Avatec (90 g/ton) as a coccidiostat.
There were no significant differences in body weight or feed conversion of broilers fed the three treatment diets at 22 days of age. However, at 49 days of age, the broilers fed Bio-Mos or BMD (5.52 and 5.68 lb) were heavier than controls (5.27 lbs, P<0.05). Likewise, efficiencies were significantly improved by the treatment diets ( Bio-Mos, 1.83;BMD, 1.82; and control, 2.01). Mortality was not significantly different between treatments at either 22 (1.25%) or 49 (5.69%) days of age. Sims (1998) and Sims et al. (1998) also reported that the feed cost per lb of meat and feed cost per bird were reduced by Bio-Mos and BMD compared to controls.
In addition, carcass market value and the return per bird less feed costs were increased by both additives. Sims and Sefton (1999) concluded that broilers fed Bio-Mos performed similarly to birds fed BMD, an observation supported by the current study.
BACTERIAL ENUMERATION
With E. coli challenge
Based on serotyping results, two of the four serotypes administered were recovered in culture (O88 and O2), and one (O2) has been reported to be pathogenic to poultry (Heller and Drabkin, 1977). There were also a number of O serotypes recovered that were not administered. These could have been introduced in previous environments the poults were exposed to such as the breeder farm or hatchery, or during transport and handling by investigators.
At day 7, significantly less total aerobic bacteria were found in intestinal samples of poults from young hens if fed the Flavomycin diet (Table 8).
Also at day 7, there were significantly fewer coliforms present in intestinal samples of poults from young hens if the poults were fed the Flavomycin diet compared to those fed Bio-Mos or Flavomycin plus Bio-Mos (Table 8). There were significantly fewer Lactobacillus sp. present at day 7 in intestinal samples of poults from young hens if the poults were fed the Flavomycin diet. Those fed the Bio-Mos diet were intermediate between those fed the Flavomycin diet and control and combination diets at day 7 and 21 (Table 8).
There were less total aerobic bacteria present in the liver samples of poults from old hens at day 7 if the poults were fed a diet supplemented with both Bio-Mos and Flavomycin (Table 8) with the Bio-Mos diet being intermediate to the control and Flavomycin diet means. Bio-Mos fed poults from young hens also had lower total aerobic counts at day 21 compared to Flavomycin or combination fed poults. Also, there were significantly fewer Lactobacillus sp. present in liver samples of poults from young hens fed a diet containing Bio-Mos or Bio-Mos plus Flavomycin at the end of week one and Bio-Mos at day 21.
Without E. coli challenge
At day 7, there were fewer total aerobic bacteria in the intestines of poults from young hens if Bio-Mos and Flavomycin were both present in the diet (Table 9) with the Bio-Mos diet being intermediate to all other treatment means.
There were significantly fewer total aerobic bacteria in intestinal samples of poults from young hens at day 21 for the Bio-Mos diet compared to those fed Flavomycin, with levels for those fed the control and combination diets intermediate (Table 9). Also, at day 21, poults from young hens had significantly fewer total aerobic bacteria in intestinal samples than poults from old hens for the control diet (5.86 vs. 6.47, Log10 values) (Table 9).
There were also fewer coliforms present at day 7 in the intestine of poults from young hens where the poults were fed a diet with Bio-Mos and Flavomycin, compared to those fed Flavomycin alone. Levels in those fed the Bio-Mos and control diets were intermediate (Table 9). At day 21, there were fewer coliforms in intestinal samples from poults of old hens if the poults were fed a diet containing Flavomycin (Table 9) compared to those poults fed the combination diet, with levels of those fed the control and Bio-Mos diets intermediate. Also, poults from old hens had fewer coliforms present than poults from young hens if fed the Flavomycin diet (4.11 vs. 5.85, Log10 values) (Table 9).
There were fewer total aerobic bacteria in liver samples at day 7 for the Bio-Mos and combination diets (Table 9) for poults from young hens. Across both hen ages, there were fewer total aerobic bacteria found in liver samples at day 7 for poults fed diets containing Bio-Mos and Bio-Mos plus Flavomycin, with levels of those fed the control diet intermediate (Table 9). There were fewer coliforms at day 7 in liver samples of poults from young hens if the poults were fed Bio-Mos.
There were also fewer coliforms in liver samples for poults from young hens if fed a diet containing Bio-Mos, compared to those fed the control diet. The levels in those fed Flavomycin and combination diets were intermediate (Table 9). Also at day 7, there were lower levels of Lactobacillus sp. in livers of poults from young hens for the combination diet, with levels of those fed the Bio-Mos diet intermediate to other treatment means (Table 9).
RELATED RESEARCH
The tisue bacteria levels observed in this study are inconsistent with respect to treatment effects, which agrees with other reports. Stanley et al. (1996) previously reported that the inclusion of Bio-Mos in the diet of broiler chicks was unable to reduce the numbers of total coliforms in the ceca. The current study found that the inclusion of Bio-Mos had intermediate effects on total coliform bacteria found in the ceca plus intestines. The current study also demonstrated no gross changes in the composition of bacteria present due to inclusion of Flavomycin. This agrees with findings by Brenes et al. (1989) who demonstrated that Flavomycin did not influence the composition of intestinal microflora of chicks, which included coliforms, lactobacilli, enterococci and Clostridia perfringens.
However, other reports suggest that Bio-Mos can alter the intestinal microflora. It was reported that pathogenic bacteria, which display mannose-specific fimbriae, can be removed from the gastrointestinal tract by introducing dietary MOS to the intestine (Newman, 1994). By decreasing the numbers of unfavorable bacteria in the intestinal lumen, the potential exists for improved animal performance since an environment suitable for the greatest nutrient utilization can be attained.
In the current study, the inclusion of Bio-Mos and Flavomycin only altered intestinal and liver bacterial populations in a transient way. By day 21, there were no major differences in intestinal or liver bacteria numbers. However, liver total aerobic bacteria numbers in poults fed Bio-Mos tended to be lower than in poults fed the control diet for poults from young hens. Intestinal coliform numbers found in unchallenged poults from old hens tended to be lower in those poults fed Flavomycin.
CONCLUSIONS
This trial has confirmed previous studies that have shown improved body weights and weight gains of poults when fed Bio-Mos or Flavomycin even in lieu of an E. coli challenge. Similar recently reported work (Sims, 1998; Sims et al., 1998; Sims and Sefton, 1999) demonstrated that the inclusion of Bio-Mos and a growth promoting antibiotic (BMD) improved growth in turkeys at 18 weeks of age, and indicated additive effects on poult performance. In conclusion, Bio-Mos is an effective and viable alternative to dietary growth promotant antibiotics for the rearing of turkeys.
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Authors: A.S. FAIRCHILD1, J.L. GRIMES1, F.W. EDENS1, M.J. WINELAND1, F.T. JONES2, and A.E. SEFTON3
1 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina,
2 Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas,
3 Alltech Biotechnology Center, Nicholasville, Kentucky, USA
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