Alternatives to the use of antimicrobial feed additives in nursery diets: A pilot study

Alternativas al uso de aditivos antimicrobianos en las dietas de destete: Un estudio piloto

Alternatives à l'usage d'additifs antimicrobiens dans le régime alimentaire des pouponnières: Une étude pilote

Darryl Ragland, DVM, PhD; Jessica L. Schneider, RVT; Sandra F. Amass, DVM, PhD, Diplomate ABVP; Michael A. Hill, BVetMed, MS, PhD, MRCVS

Department of Veterinary Clinical Sciences, Purdue University School of Veterinary Medicine, West Lafayette, Indiana. Corresponding author: Dr Darryl Ragland, Purdue University, VCS/LYNN, 625 Harrison Street, West Lafayette, IN 47907-2026; Tel: 765-494-1209; Fax: 765-496-2608.

Cite as: Ragland D, Schneider JL, Amass SF, et al. Alternatives to the use of antimicrobial feed additives in nursery diets: A pilot study. J Swine Health Prod. 2006;14(2):82-88.
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Keywords: swine, inorganic minerals, probiotics, antimicrobial resistance, enterococcus
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Received: February 24, 2004
Accepted: July 5, 2004

The use of feed-grade antimicrobials at subtherapeutic levels for growth promotion has evolved into a highly controversial issue, subjecting animal agriculture to increasing scrutiny. This use of antimicrobials is being vigorously challenged by human medical organizations and special interest groups, because they perceive that it has encouraged the emergence of antimicrobial-resistant bacteria that are being disseminated to human beings through the food chain.¹ Because a conclusive link between the practice of antimicrobial growth promotion in animals and transfer of resistant bacteria to human beings has yet to be demonstrated, the validity of such a conclusion is tenuous.² However, in the absence of scientific evidence, the vigor with which this issue is being pursued has prompted federal legislation that, if approved, would restrict the availability of antimicrobials for growth promotion. In Denmark, a ban on subtherapeutic use of antimicrobials has resulted in a near doubling of the total usage of antimicrobials for therapeutic treatment of animals that have succumbed to infectious diseases.³ Therefore, a ban on subtherapeutic antimicrobial use in swine feeding programs could exert a profoundly negative effect on the health and welfare of pigs in the United States.

The controversy surrounding antimicrobial growth promotion and demands for an immediate ban of the practice have prompted interest in alternatives to the use of antimicrobials in swine feeding programs. A plethora of products described as antimicrobial alternatives have been available for years, and some represent integral components of swine nutritional programs for nursery pigs.^4,5 However, there has been reluctance to adopt these products in swine feeding programs as sole replacements to antimicrobial growth promoters because questions persist about their ability to effectively enhance performance and suppress disease. There is also a lack of knowledge related to antimicrobial resistance patterns associated with ingestion of compounds described as antimicrobial alternatives. Therefore, this pilot study was undertaken with the intent of achieving two objectives related to the use of antimicrobial alternatives in swine feeding programs. The first objective was to evaluate pig performance in response to inorganic mineral supplementation and probiotic supplementation of swine diets. The second objective was to determine if consumption of inorganic minerals and probiotic feed supplements influence development of vancomycin resistance in enterococci.

Materials and methods

Experimental design

A 35-day growth assay was used to evaluate the effect of inorganic mineral or probiotic supplementation of nursery diets on pig performance. Pigs were assigned to treatments on entering the nursery (Day 0). The experimental design consisted of a randomized complete block design with a total of four replicate blocks. Pen constituted the experimental unit and six dietary treatments were used (Table 1).

Table 1: Nursery diet composition for pigs fed either a basal diet or the same diet supplemented with a probiotic, carbadox, copper sulfate, or zinc oxide at the expense of corn* * Phase 1 diets were fed Days 0 to 21 and Phase 2 diets Days 21 to 35. BioPlus2B (Chr Hansen Inc, Milwaukee, Wisconsin) was added to the basal diet at 0.50 kg/tonne (1.1 x 106 spores/g of feed) or 0.60 kg/tonne (1.3 x 106 spores/g of feed). Carbadox was added to the basal diet at 2.51 kg/tonne. Copper sulfate (5.2%) was added to the basal diet at 0.75 kg/tonne (192.40 g copper/tonne). Zinc oxide (72%) was added to the basal diet at 3.76 kg/tonne (2712.68 g zinc/tonne). † Choice white grease. ‡ 78.5% L-lysine. ¶ 99% methionine. § Supplied per kg of Phase 1 complete diet: vitamin A, 6105 IU; vitamin D, 611 IU; vitamin E, 44 IU; vitamin B12, 40 mg; menadione, 2.0 mg; riboflavin, 7.2 mg; d-pantothenic acid, 22.2 mg; niacin, 44 mg. Supplied per kg of Phase 2 complete diet: vitamin A, 5990 IU; vitamin D, 599 IU; vitamin E, 43.6 IU; vitamin B12, 30 mg; menadione, 2.0 mg; riboflavin, 7.0 mg; d-pantothenic acid, 21.8 mg; niacin, 43.6 mg. ** Supplied per kg of Phase 1 complete diet: copper, 11.2 mg; iodine, 0.42 mg; iron, 120 mg; manganese, 14.9 mg; zinc, 120 mg. Supplied per kg of Phase 2 complete diet: copper, 11.4 mg; iodine, 0.42 mg; iron, 122.6 mg; manganese, 15.2 mg; zinc, 122.6 mg. †† Selenium, 0.301 g/tonne. ‡‡ Natuphos 600 (BASF Animal Nutrition, Florham Park, New Jersey), 600 FTU phytase/g. NA = not applicable.

Pigs were weighed weekly and a feed record was maintained to monitor feed additions. Feeder weights were obtained on Day 21 (when the change was made from Phase 1 to Phase 2 diets), and on Day 35 (when the growth aspect of the study was terminated).

Pigs and housing

Ninety-six 17- to 20-day old crossbred pigs were assigned to the study, with equal numbers of barrows and gilts, weighing 5.94 to 5.96 kg on Day 0. Pigs selected for the study had not been treated with therapeutic antimicrobials and the dams of these pigs had not been treated with therapeutic antimicrobials during lactation. Pigs that developed medical conditions requiring antimicrobial therapy were withdrawn from the study and moved to another location for treatment to avoid potential introduction of bias with regard to measurement of antimicrobial resistance. The animal use protocol for the study was approved by Purdue University's Institutional Animal Care and Use Committee.

Pigs were housed at the Purdue University Animal Sciences Research and Education Center Swine Unit (West Lafayette, Indiana), four per pen in an environmentally controlled nursery in 1.98-m² pens with plastic-coated floors. Each pen was fitted with plywood partitions to prevent fecal contamination from adjacent pens.

Diets

Diet composition was representative of standard nursery diet formulations used by the Purdue University Animal Sciences Research and Education Center Swine Unit. An unmedicated basal diet (negative control) was formulated that met or exceeded NRC recommendations for nursery-age pigs.⁶ A medicated diet (positive control) was formulated by supplementing the basal diet formulation with carbadox at 55.12 g per tonne.

A diet containing pharmacological levels of copper was formulated by supplementing the basal diet formulation with copper sulfate (192.40 g copper per tonne), hereafter referred to as CuSO₄. A diet containing pharmacological levels of zinc was formulated by supplementing the basal diet formulation with zinc oxide (2712.68 g zinc per tonne), hereafter referred to as ZnO. The probiotic BioPlus2B (Chr Hansen Inc, Milwaukee, Wisconsin) was added to the basal diet formulation at two concentrations, 1.1 x 10⁶ or 1.3 x 10⁶ spores per g of feed, hereafter referred to as BP1 and BP2, respectively. BioPlus2B contains spores of Bacillus licheniformis and Bacillus subtilis. The level of 1.1 x 10⁶ spores per g represents the manufacturer's recommended inclusion rate for swine diets in the United States, and the level of 1.3 x 10⁶ spores per g represents the authorized inclusion rate for swine diets in the European Union.

Feed additives were added to the basal diet formulation at the expense of corn. The experimental diets were fed in meal form and were isonitrogenous and isocaloric. A phase-feeding protocol was used: the first nursery diet (Phase 1) was fed for the initial 21 days of the study and the second nursery diet (Phase 2) for the last 14 days. Diet compositions of the Phase 1 and Phase 2 basal diets are provided in Table 1, and calculated nutrient compositions in Table 2. The Phase 1 diets contained soybean oil, DL-methionine, dried whey, and select menhaden fish meal, whereas these products were eliminated from the Phase 2 diet formulations. Animal fat was added to the Phase 2 diet. The diets were prepared and bagged by the staff at the Purdue University Feed Mill.

Table 2: Calculated nutrient composition of the Phase 1 and Phase 2 basal nursery diets described in Table 1

Microbiology

Enterococci were cultured from rectal swabs from pigs and served as the sentinel organism to evaluate development of antimicrobial resistance in response to consumption of the experimental diets. Rectal swabs collected on Days 0, 21, and 35 were immersed in a commercially prepared solution (Cary-Blair transport media; Becton Dickinson, Franklin Lakes, New Jersey) and chilled on ice packs upon collection prior to transport to the laboratory. Swabs were homogenized in 0.3 mL of sterile water and a 10-mL sample streaked onto bile esculin agar plates to obtain individual colonies. Bile esculin agar plates were incubated aerobically at 35°C for approximately 24 hours. Colonies that hydrolyzed esculin in the agar were transferred to blood agar plates and incubated aerobically at 35°C for 24 hours to obtain pure colonies. As a presumptive test, the blood agar isolates were subjected to the catalase test, and isolates that demonstrated a weak reaction or no reaction were saved for definitive identification. Enterococci were identified using the AP120E biochemical analysis kit (bioMerieux, Durham, North Carolina), a system of enzymatic and fermentation reactions used for bacterial identification. The broth microdilution assay was used to determine susceptibility of enterococcus isolates to the glycopeptide antibiotic vancomycin hydrochloride (Sigma-Aldrich, St Louis, Missouri). This assay was performed according to the methods described by the National Committee for Clinical Laboratory Standards.⁷ Concentrations of vancomycin used to screen the enterococcus isolates for resistance were 0, 2, 4, 8, 16, 32, 64, 128, and 256 mg per L, which were identical to concentrations used in the microbiological laboratory of a local human medical center (Home Hospital, Lafayette, Indiana) as part of their antimicrobial resistance surveillance program. Minimum inhibitory concentrations (MIC) >= 32 mg per L were considered evidence of resistance to vancomycin.⁷

Calculations and statistical analysis

Least squares means for bodyweight, average daily gain (ADG), feed intake, feed efficiency, and MIC were calculated in SAS (SAS Institute, Cary, North Carolina) and subjected to the general linear models (GLM) procedure. The MIC results for the rectal samples collected on Days 21 and 35 were pooled for analysis. The least significant difference test was used as the mean separation procedure. For all tests, P < .05 was considered statistically significant.

Results

Bodyweight and average daily gain data are reported in Table 3, and feed consumption and feed efficiency data in Table 4. Pigs fed the diet supplemented with ZnO had the heaviest bodyweights on Days 21 and 35 (P < .05) and experienced the greatest ADG for the initial 21 days of the study (P < .05), compared to the other dietary treatments. Average daily gain was similar for pigs fed the ZnO and carbadox diets for the last 14 days of the study (P > .05), and was greatest for pigs fed the ZnO diet for the 35-day period (P < .05). On Day 21, bodyweights of pigs fed the BioPlus2B diets exceeded bodyweights of pigs fed the basal diet (P < .05), and were similar to bodyweights of pigs fed the CuSO₄ diet (P > .05). On Day 35, pigs fed both BioPlus2B diets were heavier (P < .05) than pigs fed the basal and CuSO₄ diets. In pigs fed the BioPlus2B diets, ADG was similar (P > .05) during the initial 21 days of the study and for the 35-day period. The ADG of pigs fed the BP2 diet exceeded (P < .05) that of pigs fed the BP1 diet during the last 14 days of the study. At 35 days, bodyweights of pigs fed the basal and CuSO₄ diets were similar (P > .05) and were the lowest among all dietary treatments. Pigs fed the CuSO₄ diet achieved heavier (P < .05) bodyweights on Day 21 compared to pigs fed the basal diet; however, pigs fed the basal diet experienced greater (P < .05) ADG than pigs fed the CuSO₄ diet between Days 21 and 35. The ADG was similar (P > .05) for pigs fed the basal and CuSO₄ diets over the 35-day study period.

Table 3: Least squares means of body weight and average daily gain (ADG) of nursery pigs fed basal diets (control) or the same diets supplemented with Bioplus2B (a probiotic), carbadox, copper sulfate, or zinc oxide* * Phase 1 diets were fed upon entering the nursery (Days 0 to 21) and Phase 2 diets Days 21 to 35. Supplemented diets were manufactured at the expense of corn in the basal diets. BP1 = BioPlus2B, a probiotic (Chr Hansen Inc, Milwaukee, Wisconsin), at 1.1 x 106 spores/g of feed; BP2 = BioPlus2B at 1.3 x 106 spores/g of feed. Carbadox at 55.12 g/tonne. ¶ CuSO4 = copper sulphate at 192.4 g copper/tonne. § ZnO = zinc oxide at 2712.68 g zinc/tonne. abcde Values with different superscripts within a row are statistically different (least significant difference test; P < .05).

Table 4: Least squares means of feed consumption and feed efficiency of nursery pigs fed basal diets (control) or the same diets supplemented with BioPlus2B (a probiotic), carbadox, copper sulfate, or zinc oxide* * Diets described in Table 3. abcdef Values with different superscripts within a row are statistically different (least significant difference test; P < .05).

Overall, pigs fed the ZnO diet had significantly higher (P < .05) feed intakes compared to pigs fed the other dietary treatments. Feed:gain ratio was highest for pigs fed BP1 diet and lowest for pigs fed the basal diet. Feed:gain for pigs fed the BP2 diet was similar (P > .05) to that of pigs fed the basal diet, and feed:gain was similar for pigs fed the BP2, carbadox, CuSO₄, and ZnO diets (P > .05).

Mean MIC of enterococcal isolates recovered on Day 0 and after consumption of the experimental diets on Days 21 and 35 (combined for analysis) are shown in Table 5. The MIC results for enterococci recovered on Days 21 and 35 were combined due to poor recovery of enterococci from pigs fed the BP1, CuSO₄, and ZnO diets. Enterococci were identified to the species level and in rank order, isolates consisted of Enterococcus faecium (69), Enterococcus faecalis (33), Enterococcus avium (27), Enterococcus durans (20), and Enterococcus gallinarum (1), respectively. Except for enterococci isolated from pigs assigned the BP2 diet, mean MICs on Day 0 were similar (P > .05). Even though MICs of isolates from pigs assigned the BP2 diet differed significantly from MICs of isolates from pigs fed the other diets, they still resided in the susceptible range for vancomycin. Mean MIC for the pooled Day 21 and Day 35 samples were similar (P > .05) for enterococci from pigs fed the BP1, BP2, CuSO₄, and ZnO diets. However, the MICs of pigs fed the basal and carbadox diets were greater (P < .05) than those for pigs fed the other experimental treatments. The MICs for the basal and carbadox diets both resided in the susceptible range for vancomycin. A total of nine pigs were removed from the study for welfare reasons. Seven pigs were removed due to exudative epidermitis that warranted antimicrobial therapy: one on the basal diet, one on the BP1 diet, one on the CuSO₄ diet, and four on the ZnO diet. Two pigs on the CuSO₄ diet were removed because they failed to make productive weight gains during the initial 3 weeks of the study.

Table 5: Least squares means of prefeeding and postfeeding MICs, determined by broth microdilution assay, of enterococci isolated from rectal swabs of nursery pigs fed basal diets (control) or the same diets supplemented with BioPlus2B (a probiotic), carbadox, copper sulfate, or zinc oxide* * Diets described in Table 3. Prefeeding samples were collected when pigs entered the nursery (Day 0) at 17 to 20 days of age. Postfeeding samples were collected Day 21 (when Phase 1 diets were replaced by Phase 2 diets) and Day 35. abc Values with different superscripts within a row are statistically different (least significant difference test; P < .05).

Discussion

Alternatives to the use of antimicrobials for growth promotion in animal production continue to evolve, and some that have shown promise for reducing antimicrobial usage in swine production include inorganic minerals and probiotics. In the present study, the diet supplemented with ZnO yielded the best overall performance. It was anticipated that the diet supplemented with carbadox would yield the best pig performance, since antimicrobial feed additives historically have been shown to enhance growth and feed efficiency to a much greater extent than nonantimicrobial feed additives.⁸ Our results were similar to those of other investigators who have reported that performance of nursery pigs fed ZnO matched or exceeded performance of pigs fed carbadox.⁹ Moreover, other studies have demonstrated that enhancement of growth and feed efficiency of nursery-age pigs is a consistent property of ZnO supplementation.^10,11 The exact mechanism of action of ZnO is still unknown, but stabilization of the enteric flora has been proposed.¹² In addition, systemic and local enteric effects have been proposed to account for the growth-promoting effects of zinc when fed at pharmacologic levels encompassing 2000 to 3000 ppm (ie, 2000 to 3000 g per tonne).¹³ On the basis of the feed intake data in our study, enhanced feed intake appears to account for part of the significant improvement in pig performance. The diet supplemented with CuSO₄ yielded poor responses in growth and feed efficiency. Copper sulfate has been described as an alternative to the use of antimicrobials for growth promotion in nursery diets and is described as having an antimicrobial effect,⁶ but the enhancement of pig performance does not appear to be as potent as that for ZnO.^10,11 Numerous studies have demonstrated a growth-promoting effect when copper is fed at pharmacological levels encompassing 100 to 250 ppm (ie, 100 to 250 g per tonne);^14-16 however, the results obtained in our study are consistent with negative growth and feed-intake responses reported in a series of feeding trials with nursery pigs.¹⁷ Compared to the feed intakes achieved by pigs fed the ZnO-supplemented diet in this study, it appears that reduced feed intake is partly responsible for the poor growth response obtained with the diet supplemented with CuSO₄.

In a compilation of studies with probiotics, investigators have recognized an overall benefit to feeding these products, but inconsistencies have prompted caution with respect to their use on a larger scale.⁸ Investigators in Europe found that probiotics consisting of bacillus spores had favorable effects on pig performance as well as control of enteric pathogens.^18,19 In our study, growth of pigs fed the diets supplemented with BioPlus2B lagged behind that of pigs consuming the diets supplemented with ZnO or carbadox.

Enterococci were selected for monitoring antimicrobial resistance because they are alleged to foster dissemination, from animals to humans, of genetic elements that facilitate resistance to vancomycin.²⁰ The isolates collected on Days 21 and 35 were combined for analysis due to poor recovery of enterococci from pigs fed the BP1, CuSO₄, and ZnO diets. We presume that the dietary treatments altered the enteric flora such that our ability to isolate enterococci from the fecal samples was reduced. Enrichment of samples with nutrient broth prior to plating on bile esculin agar will be employed in future studies to overcome this problem. Vancomycin was selected to screen the isolates for resistance to assess the degree of vancomycin resistance associated with enterococci obtained from nursery pigs. Vancomycin use in food animals in the United States is not permitted under any circumstances.²¹ Therefore, development of vancomycin resistance in enterococci originating from food animals reflects a mechanism exclusive of selective pressure from vancomycin use. In our study, we inferred that nursery-age pigs do not represent a significant reservoir of vancomycin-resistant enterococci. Minimum inhibitory concentrations corresponding to >= 32 mg per L were considered resistant, and the MIC for all dietary treatments clustered around 2 mg per L. We recovered a heterogeneous sample of enterococci, and E faecium constituted the predominant species (46%). Enterococcus faecium is of concern in the human health arena because it is recognized as possessing genetic traits that allow it to resist the action of vancomycin and other antimicrobials.²² However, heterogeneity of the isolates did not appear to confound the susceptibility assays. Considering the predominance of E faecium in the sample pool and clustering of MIC for all treatments around 2 mg per L, we concluded that the isolates in our study lacked the genetic traits that would promote high-level resistance to vancomycin. The MIC of 32 mg per L that was considered evidence of resistance to vancomycin was based on the standard established for humans, because an MIC for vancomycin in animals does not exist.⁶ The vancomycin concentrations mirrored those used as part of the antimicrobial resistance surveillance program in the microbiological laboratory of the local human medical center and were 0, 2, 4, 8, 16, 32, 64, 128, and 256 mg per L. In retrospect, concentrations corresponding to 0.5, 1.0, and 3.0 mg per L vancomycin should have been included in the panel to better characterize the susceptibility of the isolates. The diets containing ZnO, CuSO₄, or BioPlus2B did not promote an increase in MIC to vancomycin. However, isolates from pigs fed the basal and carbadox diets did exhibit a slight increase in MIC to vancomycin. Although this increase in MIC was inconsequential, this observation illustrates the complexity of the phenomenon of antimicrobial resistance and raises further questions regarding the influence of antimicrobial use on the expression of antimicrobial resistance. Carbadox was the only antimicrobial to which pigs were exposed in our study, and therapeutic antimicrobials were not used to treat any pigs enrolled in the study.

Nine pigs had to be removed from the study: seven developed exudative epidermitis that warranted antimicrobial therapy, and two failed to thrive after the initial 3 weeks of the study. First-parity sows comprised the majority of females in the farrowing group from which the pigs used in the study were derived. The prevalence of exudative epidermitis was attributed to inadequate immunocompetence of the parity-one females in the farrowing group to Staphylococcus hyicus. All affected pigs were relocated to another room, treated with therapeutic antimicrobials, and never returned to the nursery where the study was conducted. Also, the pens were separated by plywood partitions to prevent fecal contamination of adjacent pens by pigs that were fed different diets. Therefore, the slight increase in MIC for the basal diet is difficult to interpret, considering the lack of exposure of pigs to vancomycin. However, emergence of acquired resistance has been described in human beings that were not subjected to heavy consumption of antimicrobials, indicating that selective pressure is not always necessary to promote development of antimicrobial resistance.²³ It is within this perspective that the MIC for pigs fed the basal diet might increase over time without exposure to antimicrobials.

The results of our study indicate that nonantimicrobial feed additives may represent a viable replacement for antimicrobial feed additives in nursery diet formulations. Furthermore, our results demonstrate that BioPlus2B, carbadox, and the inorganic forms of copper and zinc fed at pharmacologic levels do not promote vancomycin resistance in enterococci.

Implications

• Zinc oxide may sustain acceptable pig performance as the sole growth promotant in nursery diets.
• Under the conditions of this study, BioPlus2B in nursery pig diets does not enhance growth or feed utilization.
• In this study, the young pig did not represent a significant reservoir of vancomycin-resistant enterococci.

Acknowledgment

Financial support for completion of the research project was provided by The National Pork Board and is gratefully acknowledged.

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