Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium

J. L. Turner, S. S. Dritz, J. J. Higgins and J. E. Minton J ANIM SCI 2002, 80:1947-1953.

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Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium1
J. L. Turner2,3, S. S. Dritz4, J. J. Higgins5, and J. E. Minton2,6
Kansas State University, Manhattan 66506

ABSTRACT: Ninety-ve pigs (initially 7.1 kg and 24 d of age) were used in a 28-d experiment to determine the effects of Ascophyllum nodosum seaweed extract (ANOD) on young pig growth performance and immune function in response to enteric disease challenge with Salmonella typhimurium (ST). Experimental treatments were arranged in a 2 4 factorial with main effects of disease challenge (control vs ST-challenge) and dietary addition of ANOD (0, 0.5, 1.0, and 2.0% of diet). Pigs were fed ANOD diets for 14 d and then challenged orally with ST or sterile media. There were no main effects of ANOD on growth performance end points, although there were signicant quadratic effects of ANOD on ADG (P < 0.04) and nal weight (P < 0.003), both being greatest at 1.0% ANOD. There was a positive linear effect of ANOD inclusion on ADFI (P < 0.07) and a negative linear effect on the gain-to-feed ratio (G/F) (P < 0.05). ST-challenge reduced ADG (P < 0.05), ADFI (P < 0.05), and G/F (P < 0.05) in the rst week following challenge. Daily estimates revealed reductions in feed intake in ST-infected pigs on d 2 to 4 following infection (P < 0.05). Rectal temperature was

increased maximally 2 d following ST-infection (P < 0.05). A disease challenge time interaction (P < 0.001) was observed for serum haptoglobin and 1-acid glycoprotein. Serum immunoglobulin M (IgM) was not inuenced by disease challenge, but IgM declined (P < 0.001) in all pigs over time. Serum immunoglobulin G (IgG) also was not inuenced by disease challenge, but IgG tended (P < 0.08) to increase over time. In vitro culture of porcine alveolar macrophages with 10 mg/ mL ANOD elevated (P < 0.05) prostaglandin E2 (PGE2) production over that of controls at 3 and 24 h of culture. There was no interleukin-10 response by porcine splenocytes cultured in vitro with 0.005, 0.05, 0.5, or 5 mg/mL ANOD. We conclude that this model of enteric disease elicits an acute phase response that is accompanied by increased rectal temperature and diminished feed intake. Furthermore, our results indicate some benecial effects of dietary ANOD on growth performance and no inuence of dietary ANOD on immune response in the presence or absence of ST-challenge. However, high ANOD concentrations are capable of activating porcine alveolar macrophages in vitro to secrete PGE2.

Key Words: Disease Resistance, Pigs, Salmonella, Seaweeds

2002 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2002. 80:19471953

Introduction
Extracts of certain seaweeds have antitumor effects in rodents, inhibiting mammary tumors (Yamamoto et al., 1977; Teas et al., 1984) and lung metastases (Itoh et al., 1995). In addition, extract of the marine algae Porphyra yezoensis activated murine macrophages, enhancing proinammatory cytokine production (Yoshizawa et al., 1993). Also, the fucoidin oligosaccharide blocked the function of the selectin family of cell adhesion molecules (Miura et al., 1996). The fucan polysaccharide, derived from ANOD, inhibits vascular smooth muscle cell proliferation in vitro (Logeart et al., 1997) and inhibits tumor growth in vitro and in vivo in mice (Riou et al., 1996). Thus, seaplant extracts demonstrate certain immunomodulatory effects. Behrends et al. (2000) reported that dietary supplementation of Ascophyllum nodosum (ANOD) to feedlot cattle during the 2 wk preceding slaughter may reduce the number of Escherichia coli

Contribution no. 01-487-J from the Kansas Agric. Exp. Stn. The authors gratefully acknowledge funding by the National Pork Producers Council on behalf of the National Pork Board and donation of the seaweed product used in the study by Acadian Seaplants Limited. The authors thank C. M. Hill and J. R. Werner for excellent technical support. 2 Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201. 3 Present address: Department of Animal and Range Sciences, North Dakota State University, Fargo 58105. 4 Food Animal Health and Management Center, Kansas State University, Manhattan 66506-5606. 5 Department of Statistics, Kansas State University, Manhattan 66506-0802. 6 Correspondence: 253 Weber Hall (phone: 785-532-1238; fax: 785532-7059; E-mail: eminton@oznet.ksu.edu). Received July 5, 2002. Accepted January 16, 2002.

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1948 Table 1. Diet compositiona (as-fed basis)

Ingredient
Corn Soybean meal (46.5% CP) Spray-dried whey Select menhaden sh meal Choice white grease Cornstarchb Monocalcium phosphate Limestone Salt Vitamin premixc Trace mineral premixd L-Lysine HCl DL-Methionine
a

Turner et al.

% of diet
49.65 28.03 10.00 4.50 3.00 2.00 1.20 0.68 0.35 0.25 0.15 0.15 0.05

Diet was formulated to contain 1.40% lysine, 0.90% Ca, and 0.79%

P.
b Ascophyllum nodosum extract replaced cornstarch to form the experimental treatments. c Provided the following per kilogram of complete diet: vitamin A, 11,023 IU; vitamin D3, 1,654 IU; vitamin E, 44 IU; menadione (menadione sodium bisulfate complex), 4.4 mg; riboavin, 9.9 mg; pantothenic acid, 33 mg; niacin, 55 mg; vitamin B12, 44 mcg. d Provided the following per kilogram of complete diet: Mn, 40 mg; Fe, 165 mg; Zn, 165 mg; Cu, 17 mg; I, 298 g; Se, 298 g.

in fecal and hide samples. Previous work suggests that feeding seaplants to swine is not harmful (Yap et al., 1982), although Grinstead et al. (2000) reported inconsistent and only incremental improvements in growth performance of healthy pigs. To our knowledge, there are no reports in the literature to suggest an antimicrobial property or immunomodulatory effect of ANOD extract when fed to domestic swine. Therefore, this study was conducted to evaluate the effect of dietary inclusion of ANOD (without conventional antimicrobials) on growth performance and measures of immune function of nursery pigs subjected to an enteric disease challenge with Salmonella typhimurium (ST). In addition, an in vitro companion study was performed to determine the optimal dose of ANOD that would activate alveolar macrophages (AMAC) and splenocytes of nursery pigs. Culture media were assayed for PGE2 and interleukin-10 (IL-10) as indicators of AMAC and splenocyte activation, respectively.

Experimental Procedures Experimental Design. The experimental protocol used in this study was approved by the KSU Institutional Animal Care and Use Committee. A total of 95 pigs (initially 7.1 kg and 24 d of age) were blocked by initial weight, equalized for sex, and randomly allotted to one of eight treatments in a 28-d study. Most treatments had six replicates (pens) with two pigs per pen; however, prior to the start of the study one pig assigned to the 0% ANOD diet and the control treatment (no disease challenge) died. Therefore, this pen only contained one pig. The eight treatments were arranged in a 2 4 factorial with main effects of disease challenge (control or ST) and dietary treatment (Table 1; 0, 0.5, 1.0, or 2.0% ANOD).

All pigs were housed under constant illumination in two similar environmentally controlled rooms, according to disease challenge. Pens contained one self-feeder and one nipple waterer to provide ad libitum access to feed and water. Before the start of the study, fecal samples were obtained and cultured to ensure that all pigs were not shedding Salmonella. Pigs were weighed and feed disappearance was measured on d 0, 7, 14, 21, and 28 to determine ADG, ADFI, and G/F. On d 14, each pig housed in the ST room (n = 48) received approximately 6 109 cfu of S. typhimurium in 10 mL of growth media. Each pig housed in the control room (n = 47) received a similar volume of sterile growth media. Rectal temperature was measured with a rapid response digital electric thermometer on one pig per pen through 7 d after challenge. Daily feed intake was also monitored through 7 d after challenge. On d 0, 7, and 14 with respect to disease challenge, serum samples were obtained from one pig per pen and analyzed for haptoglobin, 1-acid glycoprotein (AGP), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations. On d 7 and 14 after challenge, fecal samples were obtained from all pigs and cultured for the presence of Salmonella. Serum Analyses. Blood was collected and allowed to clot at room temperature and then stored at 4C overnight prior to harvest of serum by centrifugation. Serum was analyzed for haptoglobin using a colorimetric, enzymatic assay as described previously (Smith et al., 1998). Serum AGP was measured via radial immunodiffusion assay (Cardiotech Services, Inc., Louisville, KY). Serum IgG and IgM concentrations were determined with a commercially available ELISA (Bethyl Labs, Montgomery, TX). Serum was diluted 1:100,000 and 1:10,000 with assay buffer for analysis of IgG and IgM, respectively. In Vitro Experimental Design. A series of preliminary in vitro investigations indicated that concentrations of 0.1, 1, 10, or 100 g/mL ANOD in culture media did not stimulate the release of PGE2 or tumor necrosis factor- from porcine AMAC. Furthermore, these concentrations did not activate porcine splenocytes to secrete interferon or IL-10. In the present study, AMAC were collected from ve nursery pigs (approximately 60 d of age) by bronchoalveolar lavage (Chitko-McKown et al., 1991). The AMAC were plated at 1 mL of 1 106 cells/mL in RPMI-1640 media (Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT) and antibiotic/antimycotic (Life Technologies, Rockville, MD) in 24-well tissue culture plates (Costar, Cambridge, MA). After 3 h of culture (37C, 5% CO2), nonadherent cells were removed by aspiration, and the volume of media was brought to 1 mL with RPMI-treatment media. The treatments were as follows: control (RPMI only), 10 ng/mL lipopolysaccharide (10 ng/mL LPS; SigmaAldrich #L-6529, St. Louis, MO), or RPMI-supplemented with 0.01, 0.1, 1.0, or 10 mg/mL of ANOD, respectively. This was the maximum amount of ANOD that could be solubilized and sterile-ltered for use in tissue culture. At 3, 6, 12, and 24 h after treatment, AMAC culture

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media was harvested and frozen at 20C until assayed for PGE2 as previously described (Balaji et al., 2000). The limit of detection for PGE2 assay was 22.25 pg/mL. Splenocytes were collected from ve nursery pigs (approximately 60 d of age) as described elsewhere (Skjolaas, 2001). Briey, a 10-g sample of spleen was collected and ground through a wire mesh screen (Cell Dissociation Sieve-Tissue Grinder Kit #CD-1, Sigma-Aldrich, St. Louis). The splenocytes were washed free of connective tissue and erythrocytes by repeated lysis and centrifugation. Isolated splenocytes were plated at 0.5 mL of 2 106 cells/mL in RPMI-1640 supplemented with 10% FCS and antibiotic/antimycotic in 24-well tissue culture plates. Treatment media (0.5 mL) were added to the appropriate wells to give the following treatments: control (RPMI only), Concanavilin A (ConA, 10 g/mL; Amersham Pharmacia Biotech #17045001, Piscataway, NJ), and 0.005, 0.05, 0.5, or 5 mg/mL ANOD, respectively. Splenocyte culture media were harvested after 12, 24, and 48 h of culture (37C, 5% CO2) and frozen at 20C until assayed for IL-10 using a commercially available swine-specic ELISA (BioSource International, Camarillo, CA). The limit of detection of the assay was 9.1 pg/mL. Statistical Analyses. All animal data were analyzed with the PROC MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) as a 2 4 factorial in a randomized complete block design with repeated measures over time on each experimental unit (individual pens) (Littell et al., 1996). The model included terms for the xed effects of disease challenge, dietary treatment, time, and the appropriate interactions, and block was considered a random effect. Pairwise comparisons between disease challenge, dietary treatments, and(or) time means were made only when a signicant F-test (P < 0.05) for the ANOVA main effect or interaction was found using the LSD procedure. All means presented are least-square means. Chi squared analysis was used to detect differences in fecal shedding of Salmonella, on d 7 and 14, between disease challenge groups (Snedecor and Cochran, 1989). In vitro data were analyzed as a randomized complete block design within each culture time using the PROC MIXED procedure of SAS. The model included pig as a random variable and time and treatments as xed effects. Comparisons between treatment means, within culture times, were made only when a signicant Ftest (P < 0.05) for treatment was found using the LSD procedure. All means presented are least square means.

Results
There were no differences in ANOVA main effects for ADG, ADFI, or G/F among dietary treatments (Table 2). However, a positive linear effect was observed for ADFI (P < 0.07) and a negative linear effect for G/F (P < 0.05) with increasing ANOD. Quadratic effects were observed for ADG (P < 0.04) and pig end weight (P < 0.03), both being maximized at 1.0% ANOD (Table 2). As expected, a disease challenge time interaction was observed for ADG, ADFI, and G/F (P < 0.003). Before

challenge, ADG, ADFI, and G/F were similar between control and ST-challenged pigs. ST-Challenge resulted in a reduction in ADG (P < 0.05), ADFI (P < 0.05), and G/F (P < 0.05) as compared to controls during wk 3 of the study (Figure 1). However, by wk 4, ADFI did not differ between control and ST-challenged pigs. This increased ADFI for ST-challenged pigs resulted in improved (P < 0.05) ADG relative to that of controls in wk 4. On d 28, average weight of control pigs was not different from that of ST-challenged pigs (19.0 vs 18.9 kg, respectively; SEM 1.0). Daily feed intake and rectal temperature also were measured to assess acute effects of disease challenge. During the 7 d after challenge, there was a challenge time interaction for daily feed intake (P < 0.05) and rectal temperature (P < 0.05). Daily feed intake for ST-challenged pigs began to decline (P < 0.05) between 24 to 48 h after challenge (Figure 2) but returned to levels comparable to controls by 5 d after challenge. Rectal temperature (Figure 2) of control pigs did not differ during the 7 d after challenge. ST-Challenged pigs had a higher (P < 0.05) rectal temperature than controls on 0 d. Even with this unexpected difference on 0 d, STchallenge produced a marked and unmistakable febrile response. Rectal temperature in ST-challenged pigs was elevated on 1 d (P < 0.05), peaked on 2 d (P < 0.05), and returned to control levels by 4 d after challenge. However, rectal temperature was actually lower in ST-infected pigs on d 6 and 7 after challenge (P < 0.05). A challenge time interaction (P < 0.001) was observed for serum haptoglobin (Figure 3). Haptoglobin concentrations for controls declined over time, whereas haptoglobin in ST-challenged pigs was elevated (P < 0.05) on 7 d after challenge but returned to prechallenge levels by 14 d. A challenge time interaction (P < 0.001) also was found (Figure 3) for serum AGP. Concentrations of AGP in controls declined (P < 0.05) over time; however, serum AGP remained elevated on 7 d in ST-challenged pigs before declining on 14 d. Serum IgM was not inuenced by disease challenge, but IgM declined (P < 0.0001) over time. Serum IgG also was not inuenced by disease challenge, but IgG tended (P < 0.08) to increase over time (Figure 4). Serum haptoglobin, AGP, IgM, and IgG were not affected by dietary ANOD. As expected, fecal shedding of Salmonella was greater (P < 0.001) in ST (37.5% positive pens; 9/24) compared to control pens (0/24) on d 7 after disease challenge. By d 14, 16% (4/24) of ST-pens contained pigs that were shedding, whereas 1/24 pens in the control treatment were positive (4.2%). However, this difference was not signicant (P = 0.35). The single positive pen on d 14 was the result of a single pig that cultured positive. Yet retrospective evaluation of this animals rectal temperature and serum acute phase proteins revealed that none of these variables were elevated. Therefore, we were satised that biosecurity was maintained between the animal rooms and suspect that this pig did not have a subclinical infection.

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Table 2. Inuence of dietary Ascophyllum nodosum (ANOD) extract on pig growth performance from d 0 to 28
ANOD, %
Item
ADG, kg ADFI, kgc G/Fd End wt, kge
a b

0a
b

0.5a
0.45 0.68 0.68 19.2

1.0a
0.47 0.70 0.69 19.7

2.0a
0.44 0.71 0.64 18.8

SEM
0.02 0.04 0.02 1.1

0.42 0.63 0.69 18.2

Individual means represent 12 pens per treatment. Overall ANOVA main effect P = 0.18; linear P = 0.55; quadratic P = 0.04. c Overall ANOVA main effect P = 0.20; linear P = 0.07; quadratic P = 0.29. d Overall ANOVA main effect P = 0.13; linear P = 0.05; quadratic P = 0.22. e Overall ANOVA main effect P = 0.13; linear P = 0.39; quadratic P = 0.03.

In vitro stimulation with LPS caused a marked increase (P < 0.05) in PGE2 production by AMAC at 3, 6, 12, and 24 h of culture (Figure 5). Culture of AMAC with 0.01, 0.1, and 1 mg/mL ANOD extract failed to induce PGE2 synthesis above that of controls; however, 10 mg/ mL ANOD extract elevated (P < 0.05) PGE2 production over that of controls at 3 and 24 h of culture. Likewise, as expected, ConA stimulated (P < 0.05) IL-10 production by splenocytes, but there was no IL-10 response to the concentrations of ANOD extract tested (Figure 5).

Discussion Growth Performance. Grinstead et al. (2000) reported inconsistent and only minimal improvements in growth performance when healthy pigs were fed diets containing the microalgae Spirulina platensis. In the present study, we observed that ADFI tended to increase and G/F decreased in a linear manner with increasing levels of ANOD extract in the diet. This indicates that although ANOD may stimulate feed intake it has detrimental effects on feed efciency. Alternatively, the increased intake observed in pigs fed the 2% ANOD diet may have been overestimated slightly because our casual daily observations suggested these pigs wasted more feed by spillage through the slatted oor of their pens than the other treatment groups. This also would explain the decrease observed in feed conversion between the groups. Similar to previous reports (Yap et al., 1982; Grinstead et al., 2000), we found no negative impact on ADG or end weight when seaplants were fed to young pigs. In fact, the quadratic responses for ADG and pig end weight were both maximal at 1.0% ANOD, indicating that additional research may be warranted to evaluate growth responses of pigs fed diets at or near 1.0% ANOD in the absence of other dietary antimicrobials. The negative impact of ST-challenge on ADG during the week following infection is similar to results obtained by Balaji et al. (2000). However, we did not observe a negative effect on ADG during the second week (wk 4 of the study) following infection in ST-challenged pigs. This is consistent with the conclusion reached by Loughmiller (2000) that the effects of experimental enteric disease challenge with ST are limited to transient reductions in

feed intake and weight gain with little impact on future growth performance. Furthermore, Loughmiller (2000) reported that decreased nitrogen retention, during the 3 d following ST-challenge, was mainly associated with decreased feed intake and decreased apparent nitrogen digestibility during this period, and that an acute STchallenge had little long-term inuence on nitrogen retention in swine. Because all pigs infected with ST were housed in one room and control pigs in a separate yet identical room, the authors acknowledge that the effects of ST were confounded with the housing location. However, this approach was required to ensure biosecurity and prevent unintended infection of pigs in the control room. Thus, even though housing location and ST effects were confounded, we suggest that the effects of housing location were likely very minimal compared to the unmistakable pathophysiological effects of the enteric disease reported herein and previously (Balaji et al., 2000). Acute Phase Response. Our previous work using this model of enteric disease (Balaji et al., 2000) revealed that feed intake was depressed and rectal temperature was elevated through 5 d after infection. In the present study, we report similar acute reductions in feed intake and increases in rectal temperature during the 7 d following ST-challenge. We were surprised to nd that rectal temperature was elevated in ST-challenged pigs on d 0 because this occurred before disease challenge, and, up to that point, all animals had been treated alike. Moreover, we observed that rectal temperatures of ST-infected pigs were lower than controls on d 6 and 7 after infection. We did not observe this delayed hypothermia previously (Balaji et al., 2000), but this observation may reect variation in body temperature regulation as sick pigs recovered and reestablished their thermoregulatory set-point. We observed a decline in serum haptoglobin over time for controls, whereas serum haptoglobin was elevated on d 7 in ST-challenged pigs, as compared to d 0 and 14 after challenge. A similar temporal relationship was observed for serum AGP. Whereas AGP declined over time in controls, AGP in ST-challenged pigs remained elevated on d 7 before declining by d 14 after infection. In the present study, on the day of ST-challenge, pigs

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Figure 2. Daily feed intake (top panel) and rectal temperature (bottom panel) of control and treated pigs during the week following oral challenge with 6 109 cfu of Salmonella typhimurium. Asterisks denote means within day that differ between control and challenged pigs (P < 0.05). toglobin for controls was greater than their ST-challenged contemporaries on d 0, and this nding is difcult to interpret because AGP was not elevated in these pigs at this time. Serum Immunoglobulins. The fact that we did not observe an effect of ST-challenge on serum concentrations of IgG or IgM would suggest that the single acute enteric challenge with ST did not stimulate a signicant systemic humoral immune response or that such a response was not reected in serum IgG or IgM. Thus, the enteric infection was most likely contained within the gut through an effective innate immune response via phagocytic cells in the intestinal wall or through an immunoglobulin A-mediated response. The effectiveness of the mucosal immune response is borne out by the fact that, 7 d following challenge, just over 37% of the pens containing ST-treated pigs were cultured positive for Salmonella organisms. Changes in immunoglobulin concentrations over time most likely reect the active synthesis of antibodies by the pigs own immune system. Maternal IgM and IgG reach minimal levels in the piglets system by 2 and 4 wk, respectively (Hunter, 1986), and active

Figure 1. Average daily gain (top panel), average daily feed intake (middle panel), and feed efciency (bottom panel) of control pigs and pigs orally challenged (arrow) with 6 109 cfu of Salmonella typhimurium. Means without a common superscript differ (P < 0.05).

were approximately 38 d old. Therefore, the decline in AGP and haptoglobin over time in the controls may reect changes in normal concentrations of acute phase proteins associated with age. The fact that AGP and haptoglobin levels in ST-challenged pigs were higher on d 7 would suggest some inuence of enteric disease on the health status of these pigs during the 7 d following ST-challenge. We were surprised to nd that serum hap-

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increase in IgG and decrease in IgM probably reect ageassociated differences in antibody synthesis. In Vitro Effects of ANOD. The concentrations ANOD used to stimulate AMAC and splenocytes were chosen because they represented a broad range that, on the low end, were similar to those added to the feed and, on the high end, were many fold in excess of ANOD concentrations even in the 2% diet. In our opinion, the entire range of concentrations used in vitro probably exceeded the actual concentrations of ANOD achieved in the digesta of pigs, even on the 2% ANOD diet. Therefore, we feel that our in vitro studies provided a rigorous test of the ability of ANOD to directly activate immune cells in culture. Lipopolysaccharide has been shown to stimulate porcine AMAC to produce prostaglandins from arachidonic acid via the cyclooxygenase pathway (Bertram et al., 1989). The ANOD extract used in this study is reported to be a reasonably rich source of arachidonic acid (data provided by the manufacturer); therefore, we were sur-

Figure 3. Serum haptoglobin (top panel) and 1-acid glycoprotein (bottom panel) in control pigs and pigs orally challenged with 6 109 cfu Salmonella typhimurium. Means without a common superscript differ (P < 0.05).

synthesis of IgM and IgG does not begin until 2 and 5 wk, respectively (Hunter, 1986). Thus, the observed

Figure 4. Serum immunoglobulins of young pigs. Challenge with Salmonella typhimurium did not affect serum immunoglobulins, so data were pooled across control and disease challenged pigs. Within antibody isotype, means without common superscripts differ (P < 0.05).

Figure 5. Inuence of increasing levels of Ascophyllum nodosum extract or lipopolysaccharide (LPS) on prostaglandin E2 production by porcine alveolar macrophages (top panel) and interleukin-10 production by porcine splenocytes in response to the extract or concanavalin A (Con A; bottom panel) during in vitro culture. Within time in culture, asterisks denote treatment means that differ from respective controls (P < 0.05).

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prised that we did not detect a more profound inuence of ANOD extract on PGE2 production. Although ANOD extract provides an exogenous source of arachidonic acid, it is possible that ANOD extract alone is not a potent stimulus for activation of porcine AMAC. Therefore, the elevations in PGE2 at 3 and 24 h of culture may simply be due to the provision of exogenous arachidonic acid that was metabolized by constitutively expressed cyclooxygenase enzymes already present in the AMAC. Culture of porcine splenocytes with 10 g/mL ConA results in a distinct proliferative response (Hussain et al., 1981). Interleukin-10 was chosen for evaluation because it is an important type 2 cytokine that is increased in response to T-cell activation (Tizard, 1996) and that is produced in abundance by activated pig splenocytes (Skjolaas, 2001). Thus, by design, the present study revealed an increase in IL-10 production by porcine splenocytes cultured with ConA. However, we were unable to demonstrate an effect of ANOD extract on splenocyte production of IL-10. Again, this would suggest that ANOD extract is not capable of directly stimulating porcine splenocytes in vitro. Collectively, our in vitro data indicates that ANOD extract, at the concentrations fed in the current study, probably had little direct effect on gut-associated lymphoid tissue in pigs.

Implications
The results of the current study indicate that dietary inclusion of the seaweed extract Ascophyllum nodosum had minimal effects on pig growth performance. However, the signicant quadratic response in pig growth to increasing levels of the extract indicate that the extract may have a slight positive effect on pig growth, and this effect might warrant further study. However, both in vivo and in vitro results suggest that the seaweed extract had little positive effect on immune function in pigs. Furthermore, our model of acute enteric disease challenge elicited an acute phase response that was accompanied by a marked febrile response and decreased feed intake. Yet, the acute challenge caused only transient effects on growth performance that may not adequately model chronic inammation of repeated, multiple pathogens encountered by pigs in production settings.

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