J. Anim Sci. 2006. 84:2367-2373. doi:10.2527/jas.2005-564
© 2006 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Effects of cyadox and olaquindox on intestinal mucosal immunity and on fecal shedding of Escherichia coli in piglets
M. X. Ding,
Y. L. Wang,
H. L. Zhu and
Z. H. Yuan1
National Reference Laboratory of Veterinary Drug Residues, MOA Key Laboratory of Food Safety Evaluation, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
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Abstract
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A 2 x 3 factorial arrangement of treatments was used to determine the effects of olaquindox and cyadox on the intestinal mucosal immune response and on fecal shedding of Escherichia coli in Landrace x Large White barrows that had been orally given 1010 cfu of E. coli (O139:K88). Factors included 1) E. coli inoculation or no inoculation, and 2) no antimicrobial, 100 mg of olaquindox/kg, and 100 mg of cyadox/kg in the basal diet, respectively. The effects of cyadox and olaquindox were assessed in terms of fecal shedding of E. coli, the number of intraepithelial lymphocytes (IEL), immunoglobulin A-positive cells (APC) in the intestinal lamina propria, and ADG. There was no difference in the fecal shedding of total E. coli or the inoculated E. coli between olaquindox-supplemented pigs and cyadox-supplemented pigs during the experiment. However, fecal shedding of the inoculated E. coli in olaquindox- or cyadox-supplemented pigs was less (P < 0.05) than that in nonsupplemented pigs. Escherichia coli inoculation increased IEL and APC in the jejunum and ileum, but olaquindox or cyadox decreased IEL and APC (P < 0.05). Jejunal APC in cyadox-supplemented pigs was less (P < 0.05) than that in olaquindox-supplemented pigs. Escherichia coli inoculation reduced (P < 0.05) ADG, whereas the supplementations improved ADG (P < 0.01) during the experiment. Average daily gain in cyadox-supplemented pigs was greater (P < 0.05) than that in olaquindox-supplemented pigs. The data indicated that olaquindox and cyadox reduced the number of intestinal E. coli and suppressed E. coli-induced immune activation, which might be responsible for the enhanced growth that was observed.
Key Words: Escherichia coli • immunoglobulin A-positive cell • intraepithelial lymphocyte • pig • quinoxaline
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INTRODUCTION
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Postweaning diarrhea is a common disease of piglets that is characterized by diarrhea and weight loss. Although the etiology is multifactorial, enterotoxigenic Escherichia coli is associated with pathogenesis of the diarrhea. In the early 1950s, antimicrobials were used as feed additives to control diarrhea and to promote growth. Since then, studies have focused on metabolic modulation and intestinal bacterial changes that antimicrobial promoters cause. However, mechanisms by which antimicrobials enhance growth are not yet clearly elucidated. Growth promotion of antimicrobials may be due to intestinal microflora modifications, including suppression of intestinal pathogenic microorganisms (Visek, 1978
; Sarmiento and Moon, 1988; Corpet, 1999). Hathaway et al. (1990) proposed that changes in porcine intestinal microflora increases production of growth factors that function at the cellular level to regulate growth. For example, concentrations of IGF-I were increased in pigs fed antimicrobial-supplemented diets (Hathaway et al., 2003; Jenkins et al., 2004).
The immune system regulates other physiological systems. For instance, it regulates the GH–IGF-I axis (Kelley, 2004). Because the gut mucosal immune system contains one-half of the lymphocytes in the body (Mestecky and Meghee, 1987) and is directly exposed to intestinal microorganisms, alterations of the intestinal microflora may affect intestinal mucosal immunity. It is not clear if antimicrobials promote growth by influencing intestinal mucosal immunity.
Among antimicrobials, quinoxalines, olaquindox, and cyadox promote growth of piglets (Broz and Sevcik, 1979; Wang et al., 2005). To investigate immunomodulation and in vivo bacteriostasis of antimicrobial growth promoters, the objectives of this study were to determine the effects of olaquindox and cyadox on intestinal intraepithelial lymphocytes (IEL) and immunoglobulin A-positive cells (APC) and on fecal shedding of E. coli in piglets after experimental inoculation.
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MATERIALS AND METHODS
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The experimental procedures involving animals in the study were approved by the Animal Care Center, Hubei Academy of Medical Sciences.
Pigs
Landrace x Large-White barrows, weaned at 35 d of age, were selected from litters of sows whose offspring exhibited K88 susceptibility by an in-vitro, villus-adhesion assay (Sarmiento et al., 1988). It was confirmed that the piglets and their parents had no enteric diseases and had not been inoculated with any E. coli vaccines. The piglets were housed 5 per pen (3 x 2 m) in isolation rooms with cement floors and walls, where feed and water were freely available. The environmental temperature of the rooms was controlled at approximately 20°C. The piglets were fed a basal diet containing no antimicrobial for the adaptation period and entered the experiment at the age of 42 d.
Experimental Design
One hundred fifty piglets (10.3 ± 1.6 kg of average initial BW) were randomly allotted to 1 of 6 treatments with 5 replications (pens) each. A 2 x 3 factorial arrangement of treatments was employed with the following factors: 1) inoculation or control; E. coli was used as the challenge agent, and 2) no additive, 100 mg of olaquindox/kg (Hubei Zhongmu Anda Ltd., Hubei, China), and 100 mg of cyadox/kg (Institute of Veterinary Pharmaceutics, Huazhong Agricultural University, Wuhan, China) in the basal diet (Table 1; according to NRC, 1998). The treatments included 3 noninoculated groups (control group, olaquindox group, and cyadox group) and 3 E. coli-inoculated groups (E. coli-inoculated control group, E. coli-inoculated + olaquindox group, and E. coli-inoculated + cyadox group). Escherichia coli inoculums were orally administered 7 d after the diets were supplemented. The method described by Sarmiento et al. (1988) was used for E. coli inoculation. Each pig to be inoculated was given 50 mL of alkaline broth containing 1010 cfu/mL of E. coli (Serotype O139:K88, resistant to oxytetracycline; China Institute of Veterinary Drug Control, Beijing, China) via an orogastric tube. Each of the noninoculated pigs was given an equal volume of alkaline broth. The inoculated and noninoculated pigs were housed in completely separated rooms. The study was terminated on d 21 after the inoculation.
On the day (d 0) just before and on d 7, 14, and 21 after the inoculation, respectively, 5 pigs per treatment (1 randomly selected pig per pen) were anesthetized and slaughtered for intestinal samples. Body weight was determined weekly.
The Fecal Shedding Of Total E. coli and the Inoculated E. coli
Rectal fecal samples were aseptically obtained from 5 piglets per treatment (a random pig per pen on every sampling occasion) 1 d before inoculation and every 2 d thereafter for 2 wk. Each sample (1 g) was made to 5 serial, 10-fold dilutions according to the fecal E. coli concentrations in the pretest (before the experiment), and 100 µL of each dilution was spread in duplicate onto blood agar plates containing 30 mg/L of oxytetracycline and MacConkey agar plates, respectively. The plates were incubated at 37°C for 24 h. The hemolytic colonies on the blood agar plates were selected for slide aggregation assays with the specific serum against O139 (China Institute of Veterinary Drug Control) to identify whether they were formed by the inoculated E. coli. Total E. coli colonies were recognized according to their growth on MacConkey agar plates. The confirmed E. coli colonies on blood agar plates and the total E. coli colonies on MacConkey agar plates were counted. The numbers of total E. coli and the inoculated E. coli present in the undiluted rectal feces were calculated. The results were expressed as log10-transformed data.
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Measurements of Intestinal Mucosal Immunity
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Tissue Section Preparation.
A 1- to 2-cm-long cross-section of intestinal tissue was obtained from the mid-jejunum and midileum, respectively, and was lightly put into a phosphate-buffered paraformaldehyde (40 g/L) solution along with a small piece of rough paper on its chorion side to prevent curliness. After 24 h, 1-mm thick cross-sectional pieces were cut from each sample and embedded in a paraffin block. Tissues were sectioned at 4 µm, mounted on poly-lysine coated slides, de-paraffinized, and rehydrated sequentially.
Intraepithelial Lymphocytes.
Three slides from each sample were used for IEL counts. The sections were stained with hematoxylin and eosin. At 400x magnification, 10 well-developed villi were selected from 10 different fields in each section for IEL counts. The IEL numbers were determined per 100 enterocytes. The mean value of each sample was reported.
Immunoglobulin A-Positive Cells.
Another 3 slides were used for APC enumeration with a strept-avidin-biotin complex technique. Endogenous peroxidase activity was extinguished by incubation with 3% H2O2-PBS (pH 7.3, 0.01 M) for 10 min. The sections were digested with 0.1% trypsinase for 5 min at room temperature. After being washed with PBS, the sections were blocked by goat serum (1:50 dilution) at 37°C for 20 min in a humid chamber. The serum having been shaken off, the sections were reacted with 1:100 dilution of rabbit anti-swine-IgA antibody (provided by Q. Yang, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China) for 1 h. The sections were washed and incubated with biotinylated goat anti-rabbit-IgG antibody (Wuhan Boster Biological Technology Ltd., Wuhan, China) at 37°C for 20 min. After the final incubation, a peroxidase-conjugated streptavidin label was added, with diaminobenzidine used as substrate for the development of color. After development of desired staining, sections were washed and counterstained with Mayer’s hematoxylin. Negative controls for all immunohistochemical procedures consisted of exclusion of primary antibodies from tissue sections. Immunoglobulin A-positive cell counts were conducted with a light microscope connected to a video-based and computer-linked system (high resolution pathological image analysis system-1000, Wuhan Qianping Ltd., Wuhan, China) that was programmed to perform cell-count analyses. Because APC were mainly distributed in the jejunal and ileal lamina propria between glandular cavities, APC and interstitial cells (mainly lymphocytes) were counted respectively in 10 fields of the APC well-distributed lamina propria in each section examined under 400x magnification. The mean percentage of APC to interstitial cells in interstitial cells in each sample was calculated.
Statistical Analysis
Factors affecting variation of data included E. coli inoculation and supplementation. Escherichia coli and the inoculated E. coli counts were presented as log10-transformed values. Data were analyzed using the PROC GLM procedure in the SAS software package (SAS Inst. Inc., Cary, NC). Least squares analysis and 2-factor linear model with interactions were used to evaluate effects. Differences between treatment means were evaluated by a t-test after a significant F-test. Comparisons were considered significantly different if P 
0.05.
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RESULTS
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Growth Performance.
The diarrhea was developed in the inoculated pigs 2 or 3 d after inoculation and lasted 3 or 4 d. Spirit and appetite of all the pigs with diarrhea were clinically normal. Five of the pigs in the E. coli-inoculated + cyadox group and 6 of the pigs in the E. coli-inoculated + olaquindox group developed diarrhea, whereas 14 pigs in the E. coli-inoculated control group did. A few of the noninoculated pigs (2 in cyadox group, 3 in olaquindox group, and 3 in control group) developed diarrhea and were not found to shed the inoculated serotype E. coli by bacteriologic tests.
There was no E. coli x quinoxaline interaction on ADG (Table 2). Escherichia coli inoculation reduced (P < 0.05) ADG, whereas the supplementations improved (P < 0.01) ADG during the period of d 0 to 21 after the challenge. Average daily gain in the cyadox-supplemented pigs was greater (P < 0.05) than that in the olaquindox-supplemented pigs during the whole experiment.
Fecal Shedding of E. coli and the Inoculated E. coli.
The mean, log10-transformed number of total E. coli that shed after the inoculation is shown in Figure 1. In noninoculated pigs, the fecal shedding of total E. coli remained constant. The fecal shedding of total E. coli in the inoculated control group increased (P < 0.01) immediately after the inoculation and reached a maximum at d 5. Thereafter, the shedding decreased to approximately the level of the noninoculated control group at d 9. Fecal shedding of total E. coli in inoculated pigs was greater (P < 0.01) than that in noninoculated pigs at d 1, 3, 5, and 7 after inoculation. Total E. coli shedding in olaquindox- or cyadox-supplemented pigs was less than that in nonsupplemented pigs at d 3, 5, 7, 9, 11, and 13 (P < 0.05 at d 3, 5, 9, 11, and 13; P < 0.01 at d 7). There was no difference in the number of total E. coli between olaquindox-supplemented and cyadox-supplemented pigs.
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Figure 1. Mean log10 fecal shedding of total Escherichia coli (n = 5). Fecal shedding of total E. coli in inoculated pigs was greater than that in noninoculated pigs at d 1, 3, 5, and 7 after piglets were orally inoculated with E. coli (O139:K88). Total E. coli shedding in olaquindox- or cyadox-supplemented pigs was greater than that in nonsupplemented pigs at d 3, 5, 7, 9, 11, and 13. **The mean in inoculated pigs was greater (P < 0.01) than that in noninoculated pigs;  The mean in olaquindox- or cyadox-supplemented pigs was less (P < 0.05) than that in unsupplemented pigs; The mean in olaquindox- or cyadox-supplemented pigs was less (P < 0.01) than that in unsupplemented pigs.  control group,  olaquindox group,  cyadox group, • E. coli-inoculated control group,  E. coli-inoculated + olaquindox group,  E. coli-inoculated + cyadox group.
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The pattern of fecal shedding of the inoculated E. coli was similar to that of total E. coli (Figure 2). The fecal shedding of the inoculated E. coli in nonsupplemented pigs was greater (P < 0.05) than that in cyadox-supplemented pigs at d 3, 5, and 7, and that in olaquindox-supplemented pigs at d 5 after inoculation. There was no difference between the shedding of the inoculated E. coli in olaquindox-supplemented pigs and that in cyadox-supplemented pigs.
Intestinal Intraepithelial Lymphocytes.
There was no E. coli x supplementation interaction on IEL in the jejunum and in the ileum (Table 3). Due to the induction of E. coli inoculation, jejunal IEL increased at d 7 (P < 0.05) and at d 14 (P < 0.01) and remained greater (P < 0.05) at d 21, whereas ileal IEL increased (P < 0.05) at d 7 and decreased to the level of noninoculated pigs at d 21 after inoculation. The supplementations reduced (P < 0.05) IEL in the jejunum at d 14 and 21, and IEL in the ileum at d 7, 14, and 21. However, the jejunal and ileal IEL in olaquindox-supplemented pigs were not different from those in cyadox-supplemented pigs during the experiment.
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Table 3. Effects of olaquindox and cyadox on intestinal intraepithelial lymphocytes (IEL) in piglets (n = 5/group)
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Immunoglobulin A-Positive Cells.
An E. coli x quinoxaline interaction (P < 0.01) on APC in the jejunum was observed at d 14 after inoculation (Table 4). Escherichia coli inoculation increased APC in the jejunum at d 14 (P < 0.01) and d 21 (P < 0.05), and APC in the ileum at d 7, 14, and 21 (P < 0.05 at d 7 and 21; P < 0.01 at d 14). The supplementations affected APC in the jejunum (P < 0.05 at d 7; P < 0.01 at d 14 and 21) and APC in the ileum (P < 0.01 at d 7; P < 0.05 at d 14 and 21). Jejunal APC in cyadox-supplemented pigs were less (P < 0.05) than in olaquindox-supplemented pigs at d 14 after inoculation.
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Table 4. Effects of olaquindox and cyadox on immunoglobulin A-positive cells in intestinal lamina propria of piglets (n = 5/group)
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DISCUSSION
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The mode of E. coli or its endotoxin (lipopolysaccharide) challenge has been used for studying effects of dietary regimes, stressors, and medicines on immunity of weaned pigs (Sarmiento and Moon, 1988; Heugten and Spears, 1997; Jones et al., 2001). Enterotoxigenic E. coli colonizes in the swine small intestine and produces enterotoxins to stimulate the epithelial cells to secrete fluid into the lumen of the gut, thereby causing diarrhea (Gaastra and Graaf, 1982). Because diarrhea in piglets can be caused by nonbacterial factors, the intestinal colonization of E. coli needs to be assessed indirectly by the number of inoculated E. coli in rectal feces. Although olaquindox and cyadox exhibit antimicrobial activities in vitro (Shengxian et al., 2000), their bacteriostatic effects in vivo are not fully understood. In the current study, the shedding of the inoculated E. coli in olaquindox- or cyadox-supplemented pigs was less than that in unsupplemented pigs after inoculation. This indicated that olaquindox and cyadox suppressed the inoculated E. coli in vivo. On d 9, 11, and 13 after inoculation, there were 7 fecal samples in which the inoculated E. coli colonies were not isolated. The log10-transformed values for them were considered to equal zero for the convenience of statistical analyses. In fact, the log-values should be less than 2 because the detection limit was 100 cfu/g for 100 µL of the diluted solution per plate. However, this processing did not affect the shedding profile of the inoculated E. coli.
Escherichia coli with the K88 antigen adhere to and proliferate in the anterior small intestine, where normally very few bacteria are present, instead of being carried along with the normal movement of the chyme. It is this area of the intestine that is most sensitive to enterotoxic activity (Gaastra and Graaf, 1982). Tetracycline-resistant, K88+ enterotoxigenic E. coli adhere less avidly to isolated porcine intestinal epithelial cells when grown in media containing the tetracycline (Deneke et al., 1985). Sarmiento and Moon (1988) believed that antimicrobials decrease in-vitro adhesion of E. coli to mammalian cells by inhibition of formation and expression of adhesins, or induction of synthesis of functionally impaired pili. Because olaquindox and cyadox have bacteriostatic activities, they likely reduced the fecal shedding of E. coli (O139:K88) by suppressing the adhesion of the bacteria to the small intestine or its proliferation or both.
Spierenburg et al. (1988) estimated that the protective concentrations of 100 mg of olaquindox/kg and 100 mg of cyadox/kg in diets were found along the gastrointestinal tract till the entrance of the jejunum and the midjejunum, respectively, according to their minimal inhibitory concentrations against intestinal bacteria. This probably explains the results in our study that the number of total E. coli and the inoculated E. coli shed by the cyadox-supplemented pigs was less than those by the olaquindox-supplemented pigs.
Intraepithelial lymphocytes represent a very large and probably the most unusual population of lymphocytes in the body. Their numbers are influenced by the number and properties of the intestinal microorganisms. Zunjiang et al. (1997, 2001) reported that Salmonella typhi at the dose of 108 cfu/mL and Coxsackie virus at the dose of 10–12 unit/mL induced an increase in intestinal IEL and resulted in different proportion of T cells and B cells. In the current study, the reduction of IEL showed that cyadox reduced the number of intestinal E. coli. Immunoglobulin A-positive cells in the intestine produce secretory antibodies to participate in the control of pathogenic microorganisms. The study of Ahren et al. (1998) indicated that APC levels are relatively sensitive indicators of intestinal immune responses after 1 wk of E. coli oral inoculation. Pathogenic organisms stimulated the increase in APC (Wenneras et al., 1999; Yuan and Saif, 2002). In this study, the additives decreased IEL and APC in the jejunum and ileum after the challenge. Because olaquindox and cyadox reduced the fecal shedding of E. coli, this reduction was probably due to reducing stimulation of the inoculated E. coli to the intestinal immune system.
For this study, cyadox improved ADG by 19.5% (greater than olaquindox), which was similar to that reported by Broz and Sevcik (1979). The difference in ADG between the cyadox-supplemented and the olaquindox-supplemented pigs might be associated with the extent to which the antimicrobials suppressed the immune activations in the experimental pigs. Studies verified that immune system activations resulted in metabolic shifts, including the increasing utilization of glucose by peripheral tissues and the enhancing gluconeogenesis from lactate and glucogenic AA, by involved cytokines such as interferon-
, tumor necrosis factor, IL-1, and IL-2 (Grunfeld and Feingold, 1992; Heugten et al., 1994). These cytokines act on animal growth by the nutrient redistribution away from the growth process and toward support of immune system function.
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IMPLICATIONS
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Based on the large economic impact of piglets’ post-weaning diarrhea caused by enterotoxigenic Escherichia coli and the increasing fear for the potential development of antibiotic resistance, it is worthwhile to investigate growth-promoting mechanisms of quinoxa-lines (antimicrobial promoters) as alternatives for improving growth and efficiency of livestock. This experiment indicated that olaquindox and cyadox might improve the growth performance of pigs by reducing the stimulation of E. coli to the intestinal immune system and suppressing E. coli-induced immune activation.
1 Corresponding author: yuan5802{at}public.wh.hb.cn
Received for publication October 1, 2005.
Accepted for publication April 25, 2006.
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Abstract
INTRODUCTION
