Lipid metabolism and Sarcocystis miescheriana infection in growing swine

Veterinary Parasitology, 42 ( 1992 ) 41-51 Elsevier Science Publishers B.V., A m s t e r d a m

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Lipid metabolism and Sarcocystis miescheriana infection in growing swine M.D. Prickett, A.K. Prestwood ~and M. Hoenig Departments of Parasitology, and Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA (Accepted 28 August 1991 )

ABSTRACT Prickett, M.D., Prestwood, A.K. and Hoenig, M., 1992. Lipid metabolism and Sarcocystis miescheriana infection in growing swine. Vet. Parasitol., 42:41-51. Sixteen 2-month-old pigs were divided into four equal groups and infected with either 500 000, 1 000 000 or 3 000 000 sporocysts of Sarcocystis miescheriana. Four pigs served as uninfected controis. Pigs were bled weekly and serum was collected beginning 14 days prior to infection and continuing until 63 days after infection. Body fat composition, as measured by the specific gravity of the carcass, was not affected by infection. There were no significant effects of infection on serum concentrations of glucose, insulin, triglycerides, and total, high-density lipoprotein ( H D L ) and low-density lipoprotein (LDL) cholesterol. A slight depression in HDL cholesterol occurred during the acute phase of infection. Tumor necrosis factor ( T N F ) was not detected in serum from infected swine when assayed by a cytotoxicity assay using TNF-sensitive WEHI 164 clone 13 cells. Attempts to stimulate TNF production in RAW 264.7 cells with parasitic lysates gave mixed results. This study suggests that the disruption of lipid metabolism is not the primary cause of growth retardation in growing swine infected with S. miescheriana.

INTRODUCTION

Sarcocystis miescheriana (synonym Sarcocystis suicanis ) is an obligate heteroxenous coccidian with a canine definitive host and a porcine intermediate host with a worldwide distribution (Dubey et al., 1989 ). Growing pigs experimentally infected with S. miescheriana have retarded growth (Boch et al., 1980; Barrows et al., 1982; Daugschies et al., 1987, 1988a,b, 1989) which might result from a disruption in lipid metabolism (Daugschies et al., 1988a ). This disruption in lipid metabolism may be mediated by parasite-induced production of tumor necrosis factor ( T N F ) . This cytokine has been shown to have significant effects on lipid metabolism that result in tissue wasting and weight loss (Moldawer et al., 1988 ). Parasite-induced production of T N F has ~Author to w h o m correspondence should be addressed.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0 3 0 4 - 4 0 1 7 / 9 2 / $ 0 5 . 0 0

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been reported in infections with Plasmodium (Clark et al., 1990), Babesia (Wright et al., 1988), Trypanosoma cruzi (Tarleton, 1988), Trypanosoma brucei brucei (Rouzer and Cerami, 1980 ) and Leishmania (Pisa et al., 1990 ). Weight loss in cattle infected with Sarcocystis cruzi may be due, in part, to parasite-induced T N F production associated with infection (Fayer, 1988) since soluble S. cruzi proteins have induced the production of TNF in murine RAW 264.7 macrophages (Fayer et al., 1987 ). TNF, also called cachectin, inhibits the expression of some of the genes responsible for fat production and storage in adipocytes (Torti et al., 1985 ) and inhibits the differentiation of rat pre-adipocytes in vitro (Jewell et al., 1988 ). Serum from swine infected with S. miescheriana inhibited lipid accumulation by rat pre-adipocytes in vitro in a manner similar to cachectin, suggesting that disturbances of lipid metabolism via TNF production might be responsible for depressed weight gain (Jewell et al., 1988). The purpose of this study was to investigate the effects ofS. miescheriana on fat metabolism, as related to weight retardation, in growing swine. MATERIALS AND METHODS

Parasite Sporocysts of S. miescheriana were obtained as previously described (Strohlein and Prestwood, 1986) from the intestine of dogs fed pork from swine experimentally infected with S. miescheriana. Animals Sixteen pigs, 2 months of age and averaging 14 kg, were divided into four groups of four pigs each and infected per os with 500 000 (Group L), 1 000 000 (Group M) or 3 000 000 (Group H) sporocysts of S. miescheriana suspended in water. Four additional pigs served as uninfected controls. Pigs were provided with food and water ad libitum, and groups were kept in separate concrete-floored pens within the same closed building. Pigs were maintained on a typical corn-soybean grower ration with no antibiotics for the duration of the study. Pigs were bled for serum at weekly intervals, beginning 14 days prior to infection and continuing until the termination of the study 63 days after infection (d.a.i.). Carcass evaluation Surviving pigs were killed in an abattoir 63 d.a.i, and carcasses were evaluated by food inspectors. Carcasses were split in half and chilled overnight at 0°C. Then the 3/4 fat depth at the tenth rib and the specific gravity of one-

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half of each carcass were measured for estimation of the percentage of chemical fat in each animal carcass (Johnson et al., 1990). Biochemical profiles Serum concentrations of glucose, triglycerides, total cholesterol and the highdensity lipoprotein fraction of cholesterol (HDL-cholesterol) were measured according to the manufacturer's instructions using commercially available kits (Sigma, St. Louis, MO). The low-density lipoprotein fraction of cholesterol (LDL-cholesterol) was determined by subtracting the HDL-cholesterol value and one-fifth of the triglyceride value from the total cholesterol value (Friedwald et al., 1972). Insulin A radioimmunoassay for insulin, as previously described (Herbert et al., 1965 ), was performed using undiluted serum. Cell culture Both RAW 264.7 cells (ATCC TIB 71 ) and WEHI 164 clone 13 cells (Espevik and Nissen-Meyer, 1986) were cultured in RPMI- 1640 supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, endotoxin less than 1 ng m1-1 ), 1 m M sodium pyruvate (Gibco), 0.02 m M 2-mercaptoethanol and 100/zg m l - 1gentamicin (Gibco) (culture m e d i u m ). Cells were cultured in either 25 or 75 cm 2 flasks (CoStar, Cambridge, MA) at 37°C in 5% CO2: 95% air. R A W 2 6 4 . 7 cells Stimulation of T N F production by RAW 264.7 cells was conducted under conditions similar to those described by Beutler et al. ( 1985 ). Briefly, RAW cells were grown to confluence, harvested by scraping, washed three times in minimal essential m e d i u m (MEM) (Mediatech, Washington, DC, endotoxin less than 0.0015 ng m l - 1) with 25 m M HEPES (Gibco) (MEM-HEPES) and suspended in RPMI-1640 with 50 m M HEPES (RPMI-HEPES). Cells were plated at 1.5 × l0 S cells per well in a 24 well plate (CoStar 3424), incubated for 24 h and then washed three times with MEM-HEPES. Stimulants suspended in RPMI-HEPES were added to the desired concentration. Supernatant fluids were harvested 24 h later, centrifuged for 10 min at 1 5 0 0 × g and frozen at - 2 0 °C until assayed. To exclude possible T N F production induced by contaminating endotoxin, lipopolysaccharide (LPS) and parasitic pro-

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teins were suspended in RPMI-HEPES containing 7.5/tg ml-~ Polymyxin-B (Sigma), a technique previously used in a similar procedure with malarial proteins (Bate et al., 1988). Controls included medium alone, Polymyxin-B and LPS. The LPS was added at a final concentration of 1.25/zg m l - ~and all parasitic lysates were added at a concentration of 20/~g m l - 1.

Protein preparation Sarcocystis miescheriana bradyzoites were obtained from swine muscle as described previously (Farooqui et al., 1987). Cleaned intact parasites were suspended in a solution of 20 mM Tris, 40 mM NaC1, 10 mM ethylene diamine tetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl chloride, and 1 mM aprotinin, then disrupted by sonication. Large cellular debris and intact cells were removed by centrifugation at 1000×g for 20 min at 4°C. The supernatant fluid (soluble lysate or S 1 fraction) was harvested. Part of the S1 fraction was separated into soluble cytoplasmic and membrane components by differential centrifugation (Sharma et al., 1983 ). The S 1 fraction was centrifuged for 2 h at 34 0 0 0 × g to obtain a membrane fraction (P34) in the pellet. The supernatant fluid was further processed by centrifugation for 17 h at ! 10 000×g, yielding a fluid cytoplasmic soluble fraction (S110) and a cytoplasmic pelleted fraction (P 110). Protein concentrations were determined using the Bradford ( 1976 ) assay (Bio-Rad, Richmond, CA). TNF assay Levels of serum TNF were evaluated in a cytotoxicity assay using TNFsensitive WEHI 164 clone 13 cells (Espevik and Nissen-Meyer, 1986). Briefly, serum samples were diluted 1 : 10 and supernatant samples were diluted 1:2 in culture media. Samples were applied to a 96 well plate (CoStar 3596, Cambridge, MA) and serially diluted using a multichannel pipet (Flow Titertek). After WEHI cells were grown to confluence, they were removed with trypsin/ ethylene diamine tetraacetic acid (EDTA), washed three times with MEM and diluted to 600 000 cells m l - ~ in culture medium plus 2/,g m l - ~ actinomycin D (Sigma). Cells were seeded into the plates at 60 000 cells per well. The plates were incubated for 20 h at 37°C in 5% CO2: 95% air. At this time, 25 ,ul of a solution of 5 mg ml-~ 3-( 4,5-dimethylthiazol-2yl )-2,5-diphenyltetrazolium bromide (thiazol blue) tetrazolium (Sigma) were added per well. Plates were incubated for 4 h at 37°C in 5% CO2: 95% air. From each well, 150/.tl of medium were removed. Then, 100 ~tl of isopropanol acidified with 0.04% HC1 were added per well. The solution in each well was mixed vigorously with a pipet to solubilize the crystalline precipitate. Each individual serum sample was tested in at least two different assays. Serum from an endotoxin-shocked pig was used as a positive control for serum assays. Human recombinant TNF (rTNF; Genentech) was used as a positive control in assays of RAW cell supernatant fluids. The plate was read on a Bio-Rad Model

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2550 microplate reader at 570 n m and data were evaluated using Bio-Rad's microplate manager program. Values of T N F activity (U m l - 1) for each sample represents the inverse of the dilution at which 50% killing occurred.

In vivo production of swine TNF For the production of a positive T N F control, the standard method of T N F production, i.e. bacterial priming followed by LPS challenge, was used to produce swine serum with TNF-like activity. A 20 kg pig was injected i.p. with 5 mg of Propionibacterium acnes (ImmunoRegulin, ImmunoVet, Tampa, FL) in 20 ml of MEM, followed 7 days later by an i.v. dose of 7.5 mg of LPS in 10 ml MEM. Two hours after this inoculation, a blood sample was obtained and allowed to clot, then centrifuged. Serum was collected and stored at - 8 0 °C until used.

Statistical analysis Data were compared by analysis of variance using the Statistical Analysis System (SAS) General Linear Models procedure, then evaluated by Tukey's studentized range test (SAS, 1982 ); P < 0.05 was considered to be statistically significant. RESULTS

Clinical effects All of the control pigs and most of the infected pigs appeared normal throughout the study. Clinical signs associated with the acute phase of sarcocystiosis at 12-15 d.a.i., e.g. lethargy and inappetance, were limited to two pigs in Group H and one pig in Group M. All three of these pigs recovered from acute manifestations of the disease by 36 d.a.i. However, one of the pigs in Group H died 44 d.a.i, of causes unrelated to sarcocystiosis. The two other pigs in Group H became lethargic and anorectic by 36 d.a.i, and failed to gain weight. These pigs died 59 d.a.i, of congestive heart failure related to the Sarcocystis infection. Inspection of the carcasses of the surviving pigs in all groups revealed no visual signs of infection and all carcasses passed inspection for use as human food. During the course of the study, the control pigs became serologically positive for Sarcocystis, as demonstrated by Western blotting.

Carcass fat No statistically significant differences in the carcass fat composition of pigs in different groups were observed. The individual percentages of calculated

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chemical fat were as follows; for control group pigs: 35.4, 33.58, 25.32 and 10.4% (mean 26.2%); for pigs infected with 500 000 sporocysts: 32.6, 32.2, 31.6 and 21.3% (mean 29.4%); for pigs infected with 1 000 000 sporocysts: 33.2, 27.9, 20.5 and 6.3% (mean 22%); the surviving pig infected with 3 000 000 sporocysts had a calculated chemical fat value of 30.8%. The pig with 6.3% chemical fat had acute clinical signs and was the smallest pig at the end of the study.

Biochemical profiles Serum concentrations of glucose, triglycerides, cholesterol, LDL-cholesterol and insulin were unaffected by infection. A decrease in serum cholesterol and LDL-cholesterol was noted 15 d.a.i, in all groups, including the control group. Serum cholesterol concentrations in the infected groups were somewhat, but not significantly, lower than in the controls 43 d.a.i. There was a tendency for serum insulin concentrations to increase with age in both control and infected pigs. Serum HDL-cholesterol concentrations of infected groups were slightly depressed during the acute phase, but the differences were not statistically significant. The HDL-cholesterol values for the infected groups were generally lower than those for the controls following the acute phase of the infection. Individual pigs demonstrating clinical signs of acute sarcocystiosis had slightly higher serum triglyceride concentrations than other pigs at this point in the study. Other biochemical parameters were not affected in these pigs. The two pigs in Group H that died 59 d.a.i, had depressed serum glucose values of 63.3 and 51.4 mg dl-~ 58 d.a.i. Their HDL-cholesterol levels gradually declined after 36 d.a.i., from 48.2 and 43.5 mg dl- ~to 16.1 and 22.2 mg dl-~, respectively. Their LDL-cholesterol concentrations were slightly elevated. Other individual biochemical parameters noted above were not affected.

Serum TNF assays Serum TNF-like activity was only detected in one pig in Group H at 51 and 58 d.a.i. This pig died 59 d.a.i. Its TNF values were 20.5 U at 51 d.a.i, and 22.2 U at 58 d.a.i. Serum from an endotoxin-shocked pig used as a positive control had a mean value of 127 U m l - 1.

In vitro TNF production Results from separate experiments were inconsistent and no conclusions could be drawn.

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DISCUSSION

Although weight retardation has been associated with Sarcocystis infections in several domestic animals, the underlying mechanisms are not understood. Proposed mechanisms for weight reduction in calves infected with S. cruzi include TNF-induced disturbances in lipid metabolism (Fayer, 1988 ) and decreases in the growth-related hormones, growth hormone, insulin-like growth factor-1 (IGF-1) and the binding proteins of IGF (IGFBP) (Elsasser, 1988 ). Growth factor alterations have also been described in Sarcocystisinfected swine. These swine had a decrease in IGF-1 and an increase in some IGFBP during the acute phase (Prickett et al., 1992). In addition to weight, Sarcocystis may affect the quality of the carcass. Some swine carcasses have been condemned owing to the presence of lesions attributed to Sarcocystis (Greve, 1980) or have been designated 'lower quality' (Erber and Geisel, 1979). On the other hand, another study has suggested that meat quality may be improved slightly by Sarcocystis infection (Daugschies et al., 1988a). The carcasses in the present study passed the usual inspection procedures, indicating that any parasitic effects were not sufficiently extensive to cause condemnation. One investigation (Daugschies et al., 1988a) suggested that infection might disrupt fat metabolism because lean: fat ratios, back fat thickness and the fat thickness at the thirteenth rib were decreased, and water-holding capacity was increased, in infected pigs compared with controls. Not all of these differences were statistically significant. The loin fat thickness quotient was not affected. The percent carcass chemical fat of the infected and uninfected pigs in this study generally fell within or near the range of 31-51% reported in normal growing pigs (Johnson et al., 1990). Two pigs in the control group, three in the group infected with 500 000 sporocysts (Group L) and one in the group infected with 1 000 000 sporocysts (Group M) fell within this range. The individual pig in the control group with 10.4% body fat gained weight at the same rate as the other pigs in this group; the cause for its low body fat cannot be explained. All remaining pigs were slightly under the reported body fat of growing pigs, except for one pig in Group M which had 6.3% body fat. It was lethargic during the acute phase of infection and its incomplete recovery may account for the low body fat. The surviving pig in Group H, which had demonstrated visible signs of sarcocystiosis during the acute stage, had a normal fat composition of 30.8%. Lower glucose values have been associated with bovine sarcocystiosis (Prasse and Fayer, 1981 ). Serum glucose concentrations in the present study remained within the normal range of 50-130 mg dl -I reported in growing pigs (Tumbleson and Schmidt, 1988). Although the pigs that died 59 d.a.i. had glucose levels that were slightly depressed as compared with other pigs in this study, they were within reported normal values and this depression was

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probably due to their anorexia. Investigation of calves infected with S. cruzi revealed decreased plasma insulin during the acute phase; however, this decrease was attributed to a decrease in food intake rather than to parasite-related mechanisms (Fayer, 1988). Insulin levels measured in the present study were unaffected by the infection, but may have been affected by age. Hypertriglyceridemia has been observed in parasitic infections, resulting in cachexia (Rouzer and Cerami, 1980). Serum triglyceride concentrations in the present study generally were within or slightly below the reported normal range of 43-132 mg dl- ~in growing pigs (Tumbleson et al., 1976 ); however, some individual effects that were evident in pigs demonstrating clinical signs of acute sarcocystiosis may have been associated with the parasitic infection. Serum triglyceride concentrations in animals infected with Sarcocystis have not been previously reported. Serum cholesterol concentrations in all pigs generally fell within normal reported values of 50-140 mg dl- 1 (Tumbleson and Schmidt, 1988 ). Serum LDL-cholesterol concentrations were lower than the 70-85 mg dl-~ reported previously in growing pigs (Thaker and Bowland, 1988). Serum HDL-cholesterol concentrations were consistent with the 44-60 mg dl-1 previously reported in growing pigs (Thaker and Bowland, 1988). The two individuals in Group H that died 59 d.a.i, had low HDL-cholesterol and high LDL-cholesterol values 58 d.a.i, as a result of a gradual change beginning 36 d.a.i.; these may have been a result of the Sarcocystis-related chronic congestive heart failure. Serum cholesterol values have not previously been reported in animals infected with Sarcocystis. Control pigs became serologically positive for S. miescheriana owing to sporocyst contamination of the control group environment by either rodents, insects or animal handlers. Sporocysts probably passed intact through the porcine digestive tract, as noted previously in experimental infections of sheep and birds (Box and Smith, 1982; Munday, 1984/1985 ) or were regurgitated. Effects of this infection may or may not have been responsible for the decreases in cholesterol and LDL-cholesterol 15 d.a.i. Cholesterol values remained within normal ranges. The decrease in LDL-cholesterol during the acute phase in control pigs may have been related to this infection because values were lower than those seen in this group at other times. No obvious effects were noted in the other parameters studied. There was no detectable serum TNF-like activity associated with the infection. A possible role for TNF was suspected in swine sarcocystiosis since parasite-induced TNF production was suspected to play a role in bovine sarcocystiosis (Fayer, 1988). In mice infected with Plasmodium, TNF has been associated with fetal death and abortion (Clark and Chaudhri, 1988) and could possibly play a role in the abortion seen during the acute phase in Sarcocystis-infected sows (Erber et al., 1978 ). Probably, the serum TNF detected in one pig either was not associated with the parasitic infection or was the

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only parasite-stimulated serum T N F concentration high enough to be detected with this assay. The comparative cytotoxicity of porcine TNF for WEHI cells is not known and this assay may not be sensitive enough to detect porcine TNF. Serum from mice challenged with LPS, for instance, has much greater cytotoxicity for TNF-sensitive L929 cells (Tarleton, 1988) than the serum from the pig challenged with LPS in this study has for more sensitive WEHI cells. Another investigation assaying TNF-like activity in serum from endotoxin-shocked pigs has also reported similar low levels of cell cytotoxicity using L929 cells (Yang et al., 1990). Porcine rTNF has recently been produced (Pauli et al., 1989 ) and, when it becomes available, it could be used as a tool for further investigations with a specific enzyme-linked immunosorbent assay (ELISA) for porcine TNF. Bradyzoite lysates of S. cruzi induced T N F production in RAW 264.7 cells (Fayer et al., 1987 ). In our study, bradyzoite lysates stimulated T N F production in RAW 264.7 cells in some experiments; however; there was no stimulation in other experiments. Based on these results, it cannot be determined conclusively whether lysates of S. miescheriana bradyzoites stimulate T N F production in RAW 264.7 cells. The data presented here suggest that alterations in lipid metabolism are probably not associated with Sarcocystis infections in growing pigs. They suggest that the infection does not induce direct parasite effects on fat metabolism and that reduced food intake is probably primarily responsible for individual decreases. The results do not rule out the possibility that subtle disruptions in lipid metabolism, possibly mediated by TNF, may play a role in growth retardation owing to Sarcocystis infection. ACKNOWLEDGMENTS

This study was supported by a research grant from the Veterinary Medical Experiment Station, University of Georgia. Special thanks are given to Amy Beyer, Laura Johnson, Regina Roth, Xinzhuan Su, Rick Tarleton, Bill Vernadore and Ted Whitten for their assistance and advice.

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