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ANIMAL PRODUCTION |
* Departments of Animal Science and
and
Veterinary Biosciences, University of Minnesota, St. Paul 55108
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: distillers dried grain with solubles • ileitis • pig • polyclonal antibody • soybean hull
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Subtherapeutic levels have improved pig performance but often fail to prevent the disease (Gebhart et al., 1998; Schwartz et al., 1998; Winkelman, 1998). Food safety concerns over potential residue violations in meat and the risk of antibiotic-resistance in human strains of pathogenic organisms may preclude continued use of many antimicrobials.
Including distillers dried grains with solubles (DDGS) or soybean hulls in grow-finish diets may assist in preventing ileitis. Both feed ingredients are dietary sources of insoluble fiber (Shurson et al., 2000). Diets low in soluble nonstarch polysaccharides reduce the proliferation of pathogenic organisms in the gastrointestinal tract (Hampson et al., 1999). Dietary fiber increases the secretory function of the epithelium and may assist in impairing bacterial adhesion to the intestinal wall (Smith and Halls, 1968).
Providing passive immune protection to pigs by dietary inclusion of egg-based polyclonal antibodies also has great potential for improving resistance or preventing specific diseases. Improvements in growth performance and reduced mortality have been reported with dietary inclusion of spray-dried egg protein products under commercial production conditions (Kichura, 1997; Shipp et al., 1999). The objective of this study was to evaluate the effect of dietary inclusion of DDGS, soybean hulls, or a newly developed polyclonal egg antibody product specific to L. intracellularis (Camas Inc., Le Center, MN) on the ability of growing pigs to resist a L. intracellularis challenge.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Pigs, approximately 17 d of age, were blocked by sex, ancestry, and BW, and blocks were randomly allotted to 1 of 5 treatment groups: negative control (NC) corn-soybean meal diet without disease challenge, positive control (PC) corn-soybean meal diet with disease challenge, 20% DDGS diet with disease challenge (D), 5% soybean hulls diet with disease challenge (SH), or a 5% soybean hulls diet containing a polyclonal antibody product (Camas Inc., Le Center, MN) with disease challenge (PA). The polyclonal antibody product was produced by injecting laying hens several times with an antigen specific for L. intracellularis, collecting the eggs, harvesting and separating the yolk, and then spraying the resultant product onto soybean hulls. The DDGS utilized for the study was obtained from Al-Corn Clean Fuel (Claremont, MN). Animals were housed in isolation rooms, with 10 pigs per room (7.25 m2 per room, 10 rooms total) and 2 rooms per treatment group.
Experimental Diets
Pigs were allowed an acclimation period and then fed experimental diets that were formulated and analyzed as reported in the companion study (Whitney et al., 2006a). The dietary inclusion level of 5% soybean hulls was chosen because field reports indicated that clinical signs of ileitis were reduced when this level of soybean hulls was fed under commercial conditions (J. Goihl, Agri-Nutrition Services, Shakopee, MN, personal communication). The dietary crude fiber content provided by soybean hulls is equivalent to that contributed by 20% DDGS, and therefore, this level of DDGS was chosen (Table 1).
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. Actual dosage level was determined to be 6.4 x 108 L. intracellularis organisms per pig. Intestinal material was also screened and determined to be negative for the presence of other enteric pathogens, including Brachyspira species (by dark-field microscopy), viruses (by transmission electron microscopy), parasite ova (by flotation tests), Yersinia species, ß-hemolytic E. coli species, and Salmonella (by routine culture). Biosecurity procedures and cleaning and feeding schedules were developed to reduce the risk of cross-contamination across rooms, as described in the companion paper (Whitney et al., 2006a).
Data Collection
All growth performance, clinical scoring, evaluation of organism shedding, necropsies, internal organ measurements, lesion scoring, and organism quantification in intestinal tissue were as reported in the companion study (Whitney et al., 2006a).
Statistical Analysis
Analysis of variance was conducted on all data utilizing the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). Growth performance data were analyzed by room using ANOVA (2 replications per treatment). All other data were analyzed utilizing the individual pig as the experimental unit, providing 20 replications per treatment. Factors assessed for each model were treatment, room (treatment), and pig (treatment x room). Repeated measures analysis was conducted for alertness, gauntness, and diarrhea scores to account for differences over time postchallenge. Least squares means were used to compare the negative and positive control pigs in order to evaluate the effects of infected vs. noninfected pigs for the various response criteria measured. Analysis of variance was used to compare response criteria among the disease challenge treatments (PC, D, SH, and PA). When treatment differences were detected, means were separated using the LSD method.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Addition of DDGS to the diet increased the CP level, threonine, and tryptophan concentration of the diet. Total lysine concentration was slightly greater in the corn-soybean meal diets compared with other dietary treatments.
Growth Performance
One pig was removed from the experiment before completion because of health reasons unrelated to the ileitis challenge (diagnosed with Mycoplasma hyopneumoniae infection). Body weights, growth rate, feed intake, and efficiency of gain results are summarized in Table 2. Average initial pig weight was 5.6 kg. During the prechallenge period, growth, feed intake, and efficiency of gain were similar across all treatments (P 
0.44). At the time of challenge, pig weight averaged 18.4 kg and was similar among dietary treatment groups (P = 0.52).
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) but more severe than expected and previously reported using a similar challenge model (Winkelman, 1998).
Alertness, Gauntness, and Fecal Scores
Pig behavior appeared normal throughout the trial for all pigs, regardless of treatment. Unchallenged pigs remained healthy throughout the postchallenge period, as indicated by a lack of gauntness and normal fecal scores (Table 3). Fecal consistency was similar among pigs before challenge (P > 0.10), but stools were more watery during wk 1 (P = 0.08), wk 2 (P = 0.03), and wk 3 (P = 0.01) postchallenge in challenged pigs (PC) compared with NC pigs (P < 0.01), indirectly indicating clinical onset of ileitis. Additionally, PC pigs appeared to be more gaunt during wk 2 (P = 0.10) and wk 3 (P = 0.06) postchallenge compared with NC pigs. All pigs were in excellent body condition before challenge and during the first week postchallenge.
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0.19). Fecal looseness also increased with increasing time postchallenge (P < 0.01). Dietary treatment did not affect fecal consistency before challenge (P = 0.17), nor did it affect fecal consistency during the postchallenge period (P 0.18), although SH pigs appeared to have firmer stools, numerically, than challenged pigs on other dietary treatments during the last 2 wk of the study.
Including high-fiber ingredients has been shown to increase rate of passage through the gastrointestinal tract (Jorgensen et al., 1996), with increased peristaltic action attributed to the increase in passage rate. Potkins et al. (1991) also observed an increase in rate of passage of digesta by including wheat bran or oatmeal coproducts in the diet, both of which are excellent sources of insoluble fiber (Grieshop et al., 2001). Therefore, including greater fiber ingredients in the diet would be expected to increase looseness of stools in pigs.
Internal Organ Weights and Digesta Characteristics
Internal organ weights and digesta characteristics are summarized in Table 4. Infecting pigs with L. intracellularis increased heart, empty stomach, and liver weight relative to BW (P < 0.001) at the time of necropsy. Diet did not affect heart, empty stomach, or liver weights relative to BW in challenged pigs, however (P > 0.50). Weight of the small and large intestine, relative to BW, was increased in challenged pigs (P < 0.005), whereas length of the large (P < 0.001) but not small (P = 0.35) intestine was reduced by L. intracellularis challenge. Relative weight of the small intestine was decreased numerically in PA pigs compared with other challenged pigs, but diet did not significantly affect the relative weight (P = 0.14) or length (P = 0.28) of the small intestine in challenged pigs. No dietary treatment effects were observed for length of the large intestine (P = 0.78), but pigs in the D and SH treatment groups had increased large intestine weights relative to BW compared with challenged pigs fed the corn-soybean meal control diet (P < 0.05).
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0.54).
The reduced length of the small intestine observed in challenged pigs would suggest a more limited ability to digest and absorb nutrients. Additionally, increased internal organ weights (relative to BW) were observed in challenged pigs. Koong et al. (1985) indicated that a high positive correlation between visceral organ weight and heat production exists. Therefore, a decline in efficiency of gain, observed in the current study, would be expected because of increased maintenance requirements and reduced nutrient digestibility in challenged pigs.
Feeding high-fiber ingredients in swine diets has produced variable effects on internal organ mass. Results from the current study indicated no dietary effects on heart, stomach, and liver weight. Pond et al. (1988) observed increased liver, heart, and empty stomach weights (relative to BW) in young adult pigs fed a high-alfalfa diet. However, Ma et al. (2002) reported reduced liver and pancreas weights from feeding a 5% wheat bran diet. Cereal bran contains approximately 28% insoluble fiber and only 2.1% soluble fiber (Marlett, 1992), and therefore, a diet containing 5% wheat bran in replacement of corn could be expected to provide an additional 1.2% insoluble fiber but only 0.02% more soluble fiber. Distillers dried grain with solubles contains 42.2% insoluble fiber and 0.7% insoluble fiber (Shurson et al., 2000), and at a dietary inclusion level of 20% would be expected to contribute an additional 7.3% insoluble fiber, but would reduce dietary soluble fiber level by 0.2%. Soybean hulls contain greater concentrations of insoluble (75.5%) and soluble (8.4%) fiber compared with DDGS (Shurson et al., 2000) and would contribute an additional 2.7% insoluble fiber and 0.3% soluble fiber when included in the diet at a level of 5%, even though the crude fiber contribution of both DDGS and soybean hulls would be equivalent. Increases in endogenous secretion of saliva, gastric juice, pancreatic juice, and bile have been correlated with increasing fiber (soluble and insoluble) level of the diet, and may be associated with enlargement of these secretory organs (Zebrowska et al., 1983; Dierick et al., 1989; Wenk, 2001).
Feeding a diet containing soybean hulls increased small intestine weights, whereas feeding soybean hull and DDGS diets increased large intestine weights, relative to overall BW. These results support the observations of Kass et al. (1980), in which increased small intestine, cecum, and colon weights (as a proportion of BW) occurred when alfalfa meal (a source of insoluble and soluble fiber) was included in the growing pig diet. Ma et al. (2002) also reported an increase in intestinal tract weight when including 5% wheat bran in the diet as a source of insoluble fiber. Jorgensen et al. (1996) observed increased stomach, cecum, and colon weight when growing pigs were fed high fiber diets containing pea fiber and pectin. Dietary fiber of both sources would be primarily soluble vs. insoluble.
Because increased intestinal weight indicates an increase in energy and nutrient needs of the organ, providing high fiber, and especially high insoluble fiber, ingredients in the diet may be viewed negatively because it indirectly increases maintenance energy requirements in swine (Koong et al., 1985). Similarly, Jin et al. (1994) observed an increase in the rate of cellular proliferation in the jejunum and colon when feeding a diet containing 10% wheat straw. Based on an insoluble fiber content of 71.0% for wheat straw (Shurson et al., 2000), the 10% wheat straw diet would be expected to contribute an additional 6.0% insoluble fiber and slightly reduce the soluble fiber content of the diet, similar to including 20% DDGS. These results indicated an increase in intestinal cell turnover with insoluble fiber addition in the diet. Because L. intracellularis is an enteric pathogen that must invade mucosa cells intracellularly for infection, increasing cell turnover (by dietary insoluble fiber addition) may thereby shorten the time and reduce the ability of the organism to successfully colonize in mucosa cells.
Although DM concentration of digesta was not affected greatly by diet in this experiment, inclusion of insoluble nonstarch polysaccharides in the diet has been implicated with decreasing intestinal transit time and enhancing water-holding capacity of digesta (Kass et al., 1980; Low, 1985). Stanogias and Pearce (1985) reported no effect of adding soybean hulls or wheat bran at 7 to 15% of the diet on rate of passage but noted a faster rate of passage when dietary inclusion levels reached 22 to 30%. This would suggest that the levels utilized in the current study were perhaps too low to make any significant difference in digesta DM content. The lack of response of digesta pH to diet fed in the current study supports previous research by Dersjant-Li et al. (2001) indicating that increasing the nonstarch polysaccharide level of the diet (15% wheat middlings inclusion) resulted in no significant changes in pH or buffering capacity of digesta in the stomach and small intestine. Wheat middlings, however, would be expected to contribute a substantial amount of dietary soluble fiber compared with insoluble fiber.
Clinical Lesion Evaluation
Results for clinical lesion evaluation of the jejunum, ileum, cecum, and colon are presented in Table 5. One pig in the NC group had a lesion that was suspect for ileitis, but immunohistochemistry (IHC) analysis indicated that it was negative. Overall, 81% of pigs that were challenged exhibited lesions consistent with ileitis, which was much greater than lesion prevalence (63 and 59%) observed in 2 previous ileitis challenge experiments (Whitney et al., 2006a,b). Challenging pigs with L. intracellularis resulted in significant increases in lesion length, severity, and prevalence in the jejunum, ileum, colon, and overall compared with unchallenged pigs (P < 0.01).
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Antibody activity of the polyclonal antibody product at the time of feeding was not determined. However, based on clinical lesion scores, it appeared that the polyclonal antibody product was not active. The spraying process used would not be expected to damage or denature the protein in the antibody product. However, if the product was inactive, it would simply be a source of highly digestible amino acids and therefore may have encouraged pathogenic growth. This may have occurred, rather than deterring colonization of L. intracellularis. Similar polyclonal products have since been produced and marketed in a liquid form, providing more consistent positive results and maintaining antibody activity toward other enteric pathogens.
PCR and IHC Analysis
Laboratory results are summarized for fecal PCR and ileal tissue IHC in Table 6. All pigs tested negative for Lawsonia intracellularis by PCR and IHC before challenge. Additionally, all NC pigs remained negative throughout the entire postchallenge period. Challenging pigs with L. intracellularis resulted in a 98.7% detection rate of the organism in ileal tissue, indicating that nearly all pigs were successfully infected with ileitis. Diet did not affect fecal shedding, as determined by fecal PCR, or presence of L. intracellularis in the ileum, as determined by IHC. Lesion severity tended to be somewhat greater in the PA group compared with the SH group, however (P < 0.10).
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; Bertschinger et al., 1978). Providing insoluble fiber in the diet may improve the pig’s ability to resist bacterial enteric infection by increasing mucosal secretion (Smith and Halls, 1968; Zebrowska et al., 1983), reducing available substrate for pathogens (Drochner et al., 1978), reducing digesta transit time (Kass et al., 1980; Low, 1985), or increasing intestinal mucosal cell turnover rate through mechanical erosion of the mucosal surface (Jin et al., 1994; Varel and Yen, 1997). Immune protection against colonization with pathogenic E. coli has also been achieved by feeding pigs egg-based products produced by vaccinated hens (Erhard et al., 1996). Previous research examining dietary effects on colonization by other enteric pathogens, including L. intracellularis, is limited.
Comparison of 3 Disease Challenge Studies
Pig responses to ileitis challenge in this study and the 2 previous studies presented (Whitney et al., 2006a; Whitney et al., 2006b) differed considerably, regardless of dietary treatment imposed. Positive responses to dietary manipulation were observed in Study 2 (Whitney et al., 2006b; DDGS or antimicrobial regimen) but not Study 1 (Whitney et al., 2006a; DDGS inclusion) or the present trial (Study 3; DDGS, soybean hulls, or polyclonal antibody inclusion).
Ability of the pig to resist disease challenge via dietary manipulation appeared to be directly related to severity of the challenge, as reflected by growth performance. When evaluating pigs that were challenged and fed the control diet (PC) in all 3 trials, pigs from Study 2 grew 116 and 139% faster and consumed 16 and 16.5% more feed compared with pigs from Studies 1 and 3, respectively, during the postchallenge period. Challenged pigs from all 3 studies appeared to be successfully infected by the mucosal homogenate challenge model, as indicated by a nearly 100% detection level of L. intracellularis in ileal cells from challenged pigs.
Part of the difference observed in disease severity may be explained by differences in dosage level. Because actual quantification of dosage level was not possible before inoculation, dosage levels differed considerably, and all were above the goal dosage level of 1 x 108. Dosage level was closest to the goal for Study 2 (6 x 108) and Study 3 (6.4 x 108) and was approximately 2x greater in Study 1 (1.5 x 109). Work from Guedes et al. (2003) indicates that increasing the dosage level from 5.4 x 108 to 5.4 x 109, using a similar challenge model, increases mortality (0 vs. 6.3%, P < 0.05) and numerically (but not statistically) reduces ADG 28% and ADFI 14%.
Although dosage discrepancies between trials may explain a portion of the differences observed in disease severity between Study 1 and Study 2, it does not explain the increased severity observed in Study 3 compared with Study 2, when dosage levels for both trials were somewhat similar. Numerous other factors may contribute to development of ileitis. Transportation, overcrowding, extreme changes in weather conditions, and commingling of pigs from different sources have been identified as risk factors for clinical outbreaks of ileitis (McOrist, 1997; Bane et al., 1998; Dufresne, 1999). Schultz et al. (1997) suggested that other pathogenic organisms, such as Clostridium, bacteriodes, and E. coli, may exacerbate the severity of ileitis.
The inoculum used for these studies was screened and determined to be devoid of many common enteric pathogens, but effect of interaction with other organisms is unknown. Using a similar mucosal homogenate challenge model and dosage level, Winkelman et al. (2000a, b) observed mortality in untreated control groups ranging from 6.7 to 32%, considerably greater than mortality in most field outbreaks of ileitis. Actual dosage level provided appears to explain part but not all of the differences in mortality levels observed. Pure culture ileitis challenge models have been used (McOrist et al., 1993) and generally provide a more controlled and predictable infection but also reduce infection rate.
Results from this study suggest that minimal benefits of including DDGS or soybean hulls may be achieved for improving the pig’s ability to resist an ileitis challenge. Use of a polyclonal antibody product provided no benefit. These results are consistent with a previous experiment, which also resulted in more severe lesions, and therefore may indicate that diet composition has a minimal positive effect on gut health during a severe L. intracellularis infection. The inoculation dosage rate used for this study was quite successful in infecting most pigs but appeared to be greater than what may be considered a typical level during an ileitis outbreak under commercial conditions. Use of the mucosal homogenate model for challenging pigs does not provide the precision necessary to achieve a desired inoculation dosage as using a pure culture when studying dietary modification and enteric disease. Further research is necessary to refine the disease challenge model utilized for this study and previous studies and to allow further understanding of the mechanisms involved with ileitis and to determine the effectiveness of possible alternative nutritional interventions.
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Footnotes |
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2 Corresponding author: whitn007{at}umn.edu
Received for publication October 26, 2004. Accepted for publication February 7, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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