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Department of Animal Science, University of California, Davis, CA and * Finnfeeds, East Gano, St. Louis, MO
2To whom correspondence should be addressed. E-mail: kcklasing{at}ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • broiler • betaine • immunity • coccidiosis • osmolarity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). Tissues that rely on zwitterionic betaine as an osmolyte include the intestines, kidney, liver, brain and leukocytes. Osmolytes are particularly important in situations in which cellular dehydration is present because these compounds help minimize water loss despite a prevailing osmotic gradient. Changes in cell water volume can affect the metabolic state of cells. A slight increase in volume moves cells into a more anabolic state, whereas the reverse is true with loss of water (3
). Thus water balance homeostasis is an important factor for cells exposed to a variety of osmotic conditions. For example, leukocytes survey the body’s tissues and in the process are exposed to a variety of solute concentrations. Mammalian monocytes and macrophages accumulate or release betaine in response to hyperosmotic cell shrinkage or hyposmotic cell swelling, respectively, to maintain volume homeostasis. Consequently, betaine modulates cell functions including phagocytosis, NO release and inflammatory cytokine release (4
–7
). In canine kidney cells, 1 mmol/L betaine protects against apoptosis induced by hyperosmotic conditions (8
).
Intestinal parasites commonly infect vertebrates, and coccidia are prevalent pathogens of chickens. The coccidia strain Eimeria acervulina infects the duodenum and causes pathologic lesions that are visible both macroscopically and microscopically. Malabsorption and diarrhea are typical symptoms of coccidiosis. Morbidity and macroscopic intestinal lesions resulting from infection with E. acervulina are often (9
–12
), but not always (13
–15
) diminished by dietary betaine, especially when a coccidiostat is present in the diet. The mechanism for the modulating effect of betaine is not known, but does not appear to be due to direct effects on the parasite. It is possible that the action of betaine relates to its influence on phagocytes because these cells are important in protection against coccidia (16
). Alternatively, betaine may affect water balance of intestinal epithelial or connective tissue cells and subsequently change their resistance to coccidial penetration or proliferation.
The purpose of these studies was to determine the effect of betaine on the morphology of the intestinal epithelia and numbers of leukocytes drawn to the intestinal epithelium during an E. acervulina challenge. Second, we aimed to determine the interactions between betaine and osmolarity on the function of chicken macrophages and of heterophils, which are the avian equivalent of the mammalian neutrophil. We examined their response to E. acervulina by measuring phagocytosis, cytokine release and NO release. We also examined the chemotaxis of blood monocytes toward chemotactic factors released by heterophils exposed to E. acervulina.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The effects of a coccidia infection and dietary betaine on intestinal morphology were determined in growing chicks. This experiment was designed as a 3 x 2 factorial arrangement of treatments with three levels of added betaine (0.0, 0. 5 or 1.0 g/kg) and two levels of E. acervulina (- or +). Betaine (Betafin BCR, 970 g/kg Betaine, Finnfeeds, St. Louis, MO) was added to the basal diet (Table 1
) in place of cellulose to obtain the desired betaine levels. The basal diet (no added betaine) contained 0.18 g/kg betaine, 7.3 g/kg methionine and 4.4 g/kg cysteine as determined by analysis. Diets exceeded nutrient requirements for broiler chicks (17
), and the methionine plus cysteine levels were 30% in excess of the requirement, which eliminates the methionine-sparing effect of betaine. Diets were provided to 1-d-old chicks (mixed sex, Cobb x Cobb; Cobb Vantress, Siloam Springs, AR) and consumed ad libitum. Chicks were housed at seven chicks per pen and eight pens per treatment in battery brooders (Petersime Incubator, Gettysburg, OH) with thermostatically controlled heaters set at 35°C. At 14 d of age, chicks in four pens per diet were inoculated orally with 4.6 x 104 sporulated oocysts of E. acervulina AC50 (supplied as a gift from Pat Augustine, USDA, Beltsville, MD). Weight gain, feed intake and feed efficiency were determined after 14 and 21 d of feeding (i.e., before the coccidia challenge and for 1 wk after the challenge). Experiments and procedures were approved by the University of California committee on animal care and use.
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1.5 cm in length were flushed with saline and fixed in 100 g/L buffered formalin (pH 7.0). Fixed intestinal samples were sectioned, stained with hematoxylin-eosin and evaluated for the following: thickness of the lamina propria; villous height, from the base of the lamina propria to the apex of the villus; villous width at its midpoint; and crypt depth between adjacent villi. Morphometric data were collected on 10 different villi per chick on each of two different serial sections. Measurements were made and analyzed by computer-aided light microscopic analysis at magnifications between 10 and 100X using Image-Pro-Plus analysis software for the PC (Media Cybernetics, Del Mar, CA). Assessments were made only on cleanly sectioned, perpendicular villi as described previously (18
). For immunohistochemical analysis of intraepithelial lymphocytes and lamina propria leukocytes, tissue samples were submerged in cryoprotectant gel and frozen at -80°C. Tissue blocks were sectioned (6 µm) and air-dried on glass slides; the sections were fixed in acetone and redried. Sections were incubated with mouse anti-chicken CD45 monoclonal antibody (Southern Biochemical, Birmingham, AL) for 1 h and then rinsed in PBS. Sections were incubated with rabbit anti-mouse immunoglobulin tagged with peroxidase with 5 mL/L bovine serum albumin for 1 h and rinsed. Peroxidase activity was developed by incubating sections with 0.1 mL/L H2O2 and 3,3'-diamino-benzidine-tetrahydrochloride. The slides were counterstained with hematoxylin-eosin. The number of leukocytes in 10 villi per slide and the number of leukocytes in the lamina propria underneath and within these 10 villi were enumerated.
Additional intestinal samples were taken from one chick per pen at 2 d after administration of E. acervulina for evaluation of osmolarity and betaine concentrations along the intestine. Segments (5 cm) were obtained from the duodenum, the jejunum at a position midway between Meckel’s diverticulum and the entrance of the bile ducts, the ileum at a position midway between Meckel’s diverticulum and the ileum-cecal junction, and the ceca at a point midway along its length. The contents of the lumen were flushed vigorously with saline to remove the digesta. Washed segments were opened longitudinally and scraped with glass slides to obtain the mucosa. Between 1 and 2 mL of water was added to the scrapings and the suspension was homogenized (Polytron; Brinkmann Instruments, Westbury, NY). Osmolarity was determined using a freezing point osmometer (Fiske model 110; Fiske, Norwood, MA) and values were corrected for the dilution factor due to water addition to the scrapings. Betaine concentrations of the diluted samples were determined by HPLC using a cation exchange column (Ca+2) and refractive index detection (19
).
Cell culture.
Macrophages were elicited to the peritoneal cavity by an injection of Sephadex (20
). Monocytes and heterophils were obtained from the blood and purified on Histopaque 1083 (Sigma Chemical, St. Louis, MO). Monocytes and macrophages were further purified by adhesion to plastic culture plates. Cells were washed and resuspended in RPMI 1640 containing 100 mL/L serum from a chicken 7 d after administration of E. acervulina. Cultures were incubated at 41°C and 50 mL/L CO2.
Betaine concentrations used in cell culture experiments were selected on the basis of levels observed in the plasma and intestinal epithelia of chicks fed diets containing 0 or 1 g/kg betaine. Plasma levels were 0.12 and 0.51 mmol/L, respectively. The concentration of betaine in the epithelia of the intestines ranged between undetectable levels and 1.09 mmol/kg. Assuming that epithelia samples contained 800 mL/kg water, this would be equivalent to 1.35 mmol/L. Thus, we selected the following levels of betaine to test in vitro: 0.0, 0.10, 0.50 and 1.5 mmol/L. Results of the E. acervulina challenge experiment showed that the average osmolarity of blood plasma was 308 mOsmol. The lowest osmolarity found in intestinal epithelia was 684 mOsmol and the highest was 1004 mOsmol. A preliminary experiment using monocytes indicated that cells died within 1 h at osmolarities 
1200 mOsmol. Consequently, we used culture media with solute concentrations 
900 mOsmol as treatments. The osmolarity of the media was established by the addition of NaCl.
Phagocytosis of Eimeria.
This experiment was designed as a 4 x 4 factorial arrangement of treatments with four levels of betaine and four osmolarities. Macrophages and heterophils were obtained from five broiler chicks and pooled. Phagocytes were incubated in glass slide chambers (Lab-Tek II, Nalge Nunc, Naperville, IL) containing treatments (10 replications per treatment) for 6 h and then E. acervulina were added (10 per phagocyte). Slides were incubated for two additional hours, mounted, fixed, stained with hematoxylin-eosin and evaluated for phagocytosis by counting each field. Macrophages that internalized two or more E. acervulina were considered positive.
Nitric oxide and cytokine release from phagocytes.
This experiment was designed as a 4 x 4 factorial arrangement of treatments with four levels of betaine and four osmolarities. Each treatment group was replicated 10 times using cells pooled from two chicks. Macrophages or heterophils were incubated in 6-well plastic cell culture plates containing treatments for 15 min and then E. acervulina were added (10 per phagocyte). Four hours later, media were collected and nitrite levels were determined using a kit (# G4410; Sigma Chemical) and used as in indication of NO release. The amount of interleukin (IL)-6 released was determined by ELISA (Pierce-Endogen, Rockford, IL) and the amount of IL-1 released was assessed by bioassay as described previously (20
).
Chemotaxis of phagocytes.
The experiment was designed as a 3 x 3 factorial arrangement of treatments with three levels of betaine and three osmolarities. Monocytes were isolated from venous blood taken from a 5-wk-old male broiler chick. Monocytes were incubated for 3 h in media containing 0.0, 1.0 or 10.0 mmol/L betaine to permit uptake of the osmolyte; then cells (1.0 mL; 1.33 x 106 cells) were placed in 12-mm transwells containing a membrane with a 3-µm pore size (Corning Costar Cambridge, MA) that contained the appropriate level of betaine in control media (310 mOsmol). Transwells (10 per treatment group) were placed in culture plates that contained the chemoattractant (50 mL/L) plus the appropriate betaine concentrations and osmolarity (200, 300 or 400 mOsmol). The chemoattractant was comprised of conditioned media from heterophils incubated for 4 h with five E. acervulina per heterophil. Therefore, this experiment examined the capacity of monocytes to migrate into an osmotically stressful environment in response to chemotactic signals elicited by E. acervulina. Plates were incubated for 45 min at 41°C and then the transwells were removed and stained with Wright stain (Sigma Chemical) and mounted. The number of cells that crossed the membrane was enumerated in 4 fields per well at 400X.
Statistical analyses.
Data were analyzed by ANOVA for factorial arrangements of treatments and their interactions using JMP software (SAS, Cary, NC). A probability P-value < 0.05 was considered to be statistically significant and P-values between 0.05 and 0.10 were considered to be trends. P-values > 0.10 were considered to be nonsignificant. Differences between individual or group means were detected by preplanned orthogonal contrasts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) during the subsequent week compared with control chicks (P = 0.02) and they tended to have decreased feed intake (P = 0.06) and efficiency of feed utilization (P = 0.08). Dietary betaine did not affect body weight gain, feed intake or efficiency of feed utilization in the weeks preceding the challenge with E. acervulina.
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). Challenge with E. acervulina decreased plasma betaine concentrations (P 0.01), but an interaction between dietary betaine level and E. acervulina (P = 0.04) was observed because plasma betaine concentrations decreased in chicks fed 0.0 or 1.0, but not in chicks fed 0.5 g betaine/kg diet.
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). Dietary betaine at 0.5 g/kg significantly decreased the osmolarity of the duodenal epithelium compared with the 0.0 g/kg treatment. The decrease in osmolarity due to betaine tended to be more pronounced in chicks challenged with E. acervulina (P = 0.07 for betaine by E. acervulina interaction).
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and 5
). Increased numbers of lamina propria and intraepithelial leukocytes were apparent 2 d after infection with E. acervulina and generally increased with the duration of the infection. E. acervulina increased the thickness of the lamina propria and decreased the length of the villi by d 4, and this effect continued to d 7 postinfection. Dietary betaine had an interactive effect with E. acervulina on villous height 7 d after infection (P = 0.04) in which E. acervulina decreased villous height in chicks consuming 0.0 g/kg betaine, but had a minimal effect in chicks consuming 1.0 g/kg betaine. An interaction between dietary betaine and E. acervulina was also present for numbers of lamina propria (P = 0.04 on d 7) and intraepithelial leukocytes (P = 0.04 on d 4 and P = 0.05 on d 7). In general this interaction indicates that the increase in leukocyte numbers due to E. acervulina was greater at the high level of dietary betaine (1.0 g/kg) than at 0.0 g/kg.
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) was maximal in isosmotic media (310 mOsmol) and decreased by 900 mOsmol (P < 0.01). Hyposmotic media did not affect phagocytosis. Phagocytosis by macrophages was greater in media with 0.1 mmol/L betaine than in media without betaine (P = 0.05). The effect of osmolarity on release of NO in response to E. acervulina followed a similar pattern as phagocytosis (Table 7
). Heterophils incubated with 0.1 mmol/L betaine released more NO into the media compared with those incubated in the absence of betaine (P = 0.04) and a similar difference tended to occur with macrophages (P = 0.07). Macrophages released more IL-1 and IL-6 in response to E. acervulina when incubated in 600 mOsmol than in isosmotic media (Table 8
). Betaine did not influence cytokine release by macrophages.
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). The rate of chemotaxis of monocytes was greater toward hyperosmotic media than toward isosmotic media (Fig. 2)
. Media betaine concentration affected the rate of chemotaxis (P < 0.01); the average rate across all media osmolarities was highest at 10 mmol/L betaine. However, a significant betaine by osmolarity interaction (P < 0.01) indicated that 10 mmol/L betaine had a greater effect on chemotaxis into hyposmotic and isosmotic media than into hyperosmotic media.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Increasing dietary betaine levels resulted in a corresponding increase in betaine concentrations in the intestinal epithelium and in the blood. When the highest level of dietary betaine was fed, the duodenal epithelium had a level of betaine (10 mmol/L) that was more than three times higher than the level in the jejunum. Concentrations in the ileum and ceca were very low regardless of dietary betaine level. Presumably, most of the betaine was absorbed in the duodenum and jejunum with little left for absorption in the ileum and ceca. Betaine is transported by the Na+ dependent amino acid transport system A and by the Na+ and Cl- dependent betaine-
-aminobutyric acid (GABA) transporter (21
,22
). Amino acid transport system A is present in a variety of tissues including skeletal muscle (23
), kidney (22
) and intestine (24
). The betaine-GABA transporter has been found in brain, kidney, liver, heart, skeletal muscle, placenta (25
), spleen, lung (26
), monocytes and macrophages (27
), although it has not yet been documented in the intestine. Kettunen and co-workers (28
) noted the presence of Na+-dependent active transport system for betaine in the duodenum and jejunum of broiler chickens. They also found that supplementation of betaine to the diet increased the Na+-dependent component in betaine uptake as well as the total quantity taken up by the duodenum. The duodenum also had the highest osmolarity and because this tissue uses betaine for protection against a hyperosmotic environment (28
), its high concentration of betaine may also be due to retention in the cells and not just transport across the epithelium.
Coccidiosis resulted in decreased betaine concentrations in the plasma when betaine was fed at 0.0 or 1.0 g/kg diet. The lack of an effect at 0.5 g/kg betaine is difficult to explain. The effect of coccidiosis at the other two levels may be due to decreased absorption, increased retention in the epithelia to defend against increased osmolarity or increased use as a methyl donor. Coccidiosis decreases amino acid absorption (29
), and because amino acid transporters are utilized for betaine transport, it is possible that the lower plasma levels were indicative of decreased absorption. However, betaine increases nutrient digestibilities [e.g., methionine (10
), carotenoids, lysine, protein and fat (13
)] and, as shown in this study, increases villi height and presumably absorptive area. Thus it seems more likely that the increased osmolarity in the intestinal epithelium due to coccidiosis may have caused retention of betaine to help defend cell volume and subsequently resulted in less betaine for transport in the blood. Last, coccidia-challenged broilers have increased production of S-adenosylmethionine in the liver (9
). Because betaine is used as a methyl group donor in the conversion of homocysteine to methionine in the liver, it is also possible that some of the shift in plasma betaine concentration may reflect increased methylation activity by the liver.
We measured osmolarity by scraping the intestinal epithelium away from the underlying submucosa. Thus our values represent solute concentrations within the cells of the villi, as well as interstitial fluids and any luminal fluids trapped in the folds and not removed during washing. The observed value of 940 mOsmol in the duodenum is very hyperosmotic compared with normal plasma, which we found to be 309 mOsmol. The chicks consumed food ad libitum and the intestines were full of digesta; thus, the high osmolarity could be the result of active absorption of nutrients. The values that we report for the epithelium are similar to those reported by Mongin (30
) for the luminal contents. Interestingly, E. acervulina infection increased epithelial osmolarity in the duodenum and jejunum. To our knowledge, increased epithelial osmolarity due to coccidiosis has not been reported previously.
Intestinal morphology.
The amount of E. acervulina administered to chicks was low to induce a challenge that was sufficiently mild that any modulatory effect of dietary betaine could be observed histologically. Robust infections cause severe pathology and atrophy in the intestines, making evaluation of subtle changes difficult. In our model, E. acervulina clearly caused morbidity in chicks as indicated by decreased growth rates, but we did not observe mortality or hemorrhages in the duodenum, indicating that the severity of the challenge was mild (31
). Leukocyte infiltration was increased by d 2 after challenge, indicating an active infection in the duodenum. By d 4 after challenge, the height of the villi in the duodenum was decreased and the thickness of the lamina propria was increased. These observations are typical of a coccidia infection (32
).
The shortening of duodenal villi due to E. acervulina was less severe in chicks fed 1.0 g/kg betaine than in those fed an unsupplemented diet. Similarly, 1 g/kg betaine improved the jejunal crypt:villus ratio of broilers challenged with E. maxima (33
). Augustine et al. (11
) found that dietary betaine moderates macroscopic lesion caused by either E. acervulina or E. tenella infections. Dietary betaine also decreases the invasiveness of E. acervulina and E. tenella as indicated by the number of sporozoites present in the intestinal epithelium 6 h after challenge (11
). In vitro, betaine lacks negative effects on the ultrastructure of sexual stages of E. acervulina and probably does not exhibit its protective effect by decreasing the virulence of the pathogen (10
,11
). We found that chicks fed dietary betaine had more leukocytes in the epithelium and in the lamina propria during an E. acervulina infection than those fed diets without betaine. This could result in more effective clearance of sporozoites and account for the decreased numbers in the epithelium observed previously (11
). Increased numbers of leukocytes in response to E. acervulina suggest that betaine either increased the chemotactic signals responsible for recruitment of leukocytes to the villi or had a direct effect on the leukocyte’s responsiveness to the signals.
Phagocyte function.
Phagocytes orchestrate a local inflammatory response that is important in early protection against coccidia; thus, we examined the effect of betaine on the functionality of macrophages and heterophils. The intestinal epithelia experience hyposmotic conditions after water consumption and hyperosmotic conditions during digestion, leading us to examine phagocyte function across a broad range of osmolarities. The osmolarity of the media affected all of the parameters of phagocyte function that we examined, with high osmolarity being inhibitory towards phagocytosis and NO release. Betaine did not affect the release of IL-1 or IL-6, but it enhanced phagocytosis of E. acervulina by macrophages and NO release from heterophils and macrophages. Phagocytosis and NO are critical effector functions in defense against parasites, including coccidia (34
).
Monocytes from peripheral blood had a greater rate of chemotaxis in the presence of 10 mmol/L betaine compared with 0.0 mmol/L betaine, especially when the medium was isosmotic or hyposmotic (Fig. 2)
. During an infection, monocytes leave the blood in response to chemokines and mature into macrophages. Interestingly, the rate of monocyte chemotaxis was considerably greater when they were entering into hyperosmotic media (400 mOsmol) than when they were migrating into isosmotic media. It is possible that we did not see an effect of betaine in hyperosmotic media because the rate of chemotaxis was already maximized and any additional affects of betaine could not be realized.
These studies demonstrated a modulatory effect of dietary betaine on the pathogenesis of E. acervulina infection. Betaine ameliorated the blunting of villi caused by infection and increased the numbers of intraepithelial and lamina propria leukocytes. At least part of the protective effect of betaine could be attributed to enhancement of monocyte chemotaxis and NO production by heterophils and macrophages.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 14 November 2001. Initial review completed 4 January 2001. Revision accepted 29 March 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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