Effects of the Ergot Alkaloids Dihydroergotamine, Ergonovine, and Ergotamine on Growth of Escherichia Co/I 0157: H7 in Vitro

FOODBORNE PATHOGENS AND DISEASE Volume 5, Number 5. 2008 Co Mary Ann Liebert, Inc. DOl: 10.10891/fpd.2007.0067

Effects of the Ergot Alkaloids Dihydroergotamine, Ergonovine, and Ergotamine on Growth of Escherichia co/i 0157: H7 In Vitro

M.L. Looper, 1 T.S. Edrington 2 A.S. Moubarak, 3 T.R. Callaway, 2 and C.F. Rosenkrans, Jr.3

Abstract

A series of experiments were conducted to evaluate the effects of ergot alkaloids (dihydroergotamine, ergonovme, and ergotamine) on E. coil 0157:117 in both pure and mixed ruminal fluid culture. Alkaloids were added to solutions of E. co/i 0157:H7 strains 933 (pure and ruminal cultures) and 6058 (ruminal culture only), and growth rates and colony-forming units (CFU) of E. co/i 0157:H7 were measured. Two mixtures of all three alkaloids at either 2 or 500 pM for each alkaloid decreased (p < 0.001) the growth rate of F. coli 0157:H7 in pure culture compared to the individual alkaloids. Di hydroergota mine tended (p=O.O7) to reduce growth rate of E. co/i 0157:H7 in pure culture compared with ergonovine or ergotamine alone. Increased concentrations of dihydroergotamine and ergotanhine decreased (p < 0.003) growth rate of E. co/i 0157:H7 but increasing concentrations of ergonovine did not influence ( p >0.10) E. co/i 0157:H7 growth rate. Similar to results in pure culture, a mixture of all three alkaloids at various concentrations for each alkaloid decreased (1)<0.001) the CFIJ of E. co/i 0157:H7 strain 6058 in mixed ruminal culture compared to the individual ergot alkaloids. Dihydroergotaniine decreased (p = 0.04) CFU of E. co/i 0157:1-17 strain 6058 when compared to ergonovinc but CFU were similar (p >0.10) between dihvdroer- gotamine and ergotaminc. Ruminal and (or) intestinal populations of E. co/i 0157:H7 may be influenced in livestock consuming endophyte-infected tail fescue, and these alterations could be due to the presence of ergot alkaloids in fescue plants.

Introduction could shed bacteria during the feedyard phase and (or) harvest. HE MAJORITY OF OUTBREAKS of Esc/ieric/iia Ruminants grazing pastures in the south- T co/i 0157:H7 in humans have resulted from eastern United States are likely to consume tall foods that originated from cattle, usually fescue [Festuca aru udi iacea Schreh.; syn. Lolium ground beef (Rangel etal., 2005). Ruminants that arundinaceuni (Schreb.) Darbysh] at some point shed this pathogen in their feces typically ap- during their lifetime since more than 20 million pear asymptomatic (Gansheroff and OBrien, ha of tall fescue are grown throughout this re- 2000). While a majority of research investigating gion (Bouton, 2000). It is estimated that approx- E. co/i 0157:H7 shedding has been conducted on imately 70% of this tall fescue is infected with an confined, grain-fed cattle, grazing cattle are in- endophytic fungi (Neottp/iodiu in coeno pu ia/il ni; fected with foodborne pathogens (Laegreid etal., Shelby and Dalrymple, 1987), which produce 1999; Dunn et al., 2004; Looper et al., 2006) and numerous ergot alkaloids (Bacon et al., 1977).

Dale Bumpers Small Farms Research Center, Agricultural Research Service, U.S. Department of Agriculture, Booneville, Arkansas. 2 Food and Feed Safet y Research Unit, AgriculEiral Research Service, U.S. Department of Agriculture, College Station, Texas. LT O,versitv of Arkansas, F.ivetteville, Arkansas.

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600 LOOPER ET AL.

Cattle grazing endophyte-infected tall fescue and incubated at 39 C for 24h. Two addi- have increased body temperature during sum- tional treatments were tested: 1) a mixture of all mer months, reduced reproductive performance three alkaloids at 500 1iM each (0.1 rnL of 500 j.iM and growth rate, and decreased milk produc- dihydroergotamine, 0.1 mL of 500 tM ergono- tion (Hoveland et al., 1983; Paterson et al., 1995). vine, and 0.1 mL of 500 tM ergotamine for the Effects of the consumption of tall fescue on fecal 500 tiM mixture), and 2) a mixture of all three shedding of F. coli 0157:H7 in naturally infected alkaloids at 2 tM each (0.1 mL of 2 tiM dihy- cattle has not been consistent. Fecal shedding of droergotamine, 0.1 mL of 2 iM ergonovine, and E. coil 0157:H7 was either decreased (Looper 0.1 mL of 2 tiM ergotamine for the 2 iM mixture). et al., 2003) or not altered (Looper et al., 2006) in Growth rate of E. coil 0157:H7 strain 933 (nali- cattle grazing endophyte-infected tall fescue. dixic acid resistant; maximal specific growth Short-term feeding of endophyte-infected tall rates [MSGR; cell density per hour]) were esti- fescue seed diets tended to increase fecal shed- mated via measurement of optical density (OD) ding of F. coli 0157:H7 in experimentally inoc- at 600 nm using a Spectronic 20D spectropho- ulated ewes (Looper et al., 2007). Causative tometer (Thermo Electron Corp., Madison, WI). factors associated with alterations of patho- Cell density readings were recorded every 30 genic shedding in feces of cattle consuming minutes until the OD reached 0.6. endophyte-infected tall fescue have not been elu- cidated. Therefore, a series of experiments were Mixed cultures conducted to evaluate the effects of three ergot alkaloids found in endophyte-infected tall fescue Rurninal fluid was obtained from fistulated (dihydroergotamirie, ergonovine, and ergota- Holstein cows maintained on a bermudagrass mine) on the growth of E. coil 0157:H7 in both hay (70%) and concentrate diet. Ruminal fluid pure and mixed ruminal fluid culture. was transferred anaerobically to a medium containing (per liter): 292mg of K2HPO4 . 3 Materials and Methods H20, 240 mg of KH2PO4, 120 mg of (NH4)2SO4, 480 mg of NaCl, 100 mg of MgSO4 . 71-120, 64 mg Ergot alkaloids of CaC12 2H20, 600 mg of cysteine hydrochlo- Dihydroergotamine, ergonovine, and ergota- ride, 4 g of Na2CO3, and 1 g each of sigmacell, mine were purchased from Sigma Chemical Co. glucose, xylose, and cellobiose. The final con- (St. Louis, MO). Stock solutions for each concentration of ruminal fluid was 33% (vol/vol). centration of each alkaloid were prepared in To this ruminal fluid medium, 2 m of a 1:10 0.1 mL of methanol. Each alkaloid was added to dilution of the bacteria was added (initial bac- autoclaved methanol (100%) in sealed anoxic terial concentrations were approximately 106 tubes to achieve final concentrations of 1000, colony-forming units [CFU]/mL of E. coii). One- 500, 250, 125, 62.5, 31.5, 16, 8, 4, 2, and 01.iM. milliliter aliquots of inoculated ruminal fluid Two experiments (pure cultures and mixed were serially diluted (in 10-fold increments) in cultures) were conducted, and the experimental phosphate-buffered saline (PBS, pH 7.0), plated, designs used for testing the effects of ergot al- and incubated overnight at 37C for direct kaloids on E. coil 0157:H7 are presented in the counting to determine initial bacterial concen- following sections. trations. Eschericlua coli 0157:H7 strain 933 was plated on MacConkeys agar supplemented with 20 j.ig/mL novobiocin and 25 ig/mL nali- Pure cultures dixic acid, and strain 6058 (rifarnpicin resistant) Escherichia co/i 0157:H7 strain 933 (nalidixic was plated on MacConkeys agar supplemented acid resistant) was added (0.1 mL) to 2.8 mL with 25 tg/mL rifampicin. Ten milliliters of the of autoclaved anoxic tryptic soy broth (TSB). above ruminal fluid medium was added to an- Individual alkaloids were then added (0.1 mL of oxic Balch tubes flushed with 0 2-free CO2 and stock solution) to achieve the desired final con- the tubes sealed with butyl rubber stoppers and centration (0, 2, 4, 8, 16, 31.5, 62.5, 125, 250, 500, aluminum crimps. Individual alkaloids at con- and 1000 1iM). The tubes were sealed, vortexed, centrations of 0, 2, 4, 8, 16, 31.5, 62.5, 125, 250,

ERGOT ALKALOIDS AND GROWTH OF E. COLI 0157:H7 601

TABLE 1. INFLUENCE OF THE MAIN EFFECTS OF Eicor ALKALOID AND VARYING CONCENTRATIONS OF ERGOT ALKALOID ON THE GROWTH OF E. COLI 0157:117 STRAIN 933 IN PURE CULTURE

Ergot alkaloid Maximal specific growth rate per hour

Dihyd roergotanine 0.88 ± 0.07b

1.08 ± 0.07a Ergonovine Ergotamine 1.08±O.07a Mixture of three alkaloids" 0.16 ± DiSc Concentration (pM) 2 0.91± 0.11a 4 0.87 ± 0.14ab 8 0.99 ± 0.14a 16 0.77 ± 0.14ab 31.5 0.94 ± 0.14a 62.5 0.90 + 0.14a 125 0.97 ± 0.14a 250 0.71 ±0.l4ab 500 0.56 ± 0.11bc 1000 0.35 + 0.14c

Maximal specific growth rate values followed by the different letters are significantly different at p <0.10. Dihydroergotamine, ergonovine, and ergotanhine mixture at either 2 (0.1 mL of 2 iM dihydroergotamine, 0.1 ml of 2 .tM ergonovine, and 0.1 niL of 2 tiM ergotamine) or 500 M (0.1 mT. of 500 1iM dihvdroergotaminc, 0.1 mL of 500 iM ergonovine, and 0.1 ml of 500 pM ergotamine), independent of ergot alkaloid concentration.

500, and 1000 uM and six separate mixtures of Results all three alkaloids at concentrations of 2, 8, 31.5, Pure culture 125, 500, and 1000 tM for each alkaloid were added (0.1 mL of each alkaloid) and tubes were A mixture of all three alkaloids at either 2 or incubated for 24 hours at 37G. For the six 500 tiM of each alkaloid decreased (p

602 LOOPER El AL. tration, independent of ergot alkaloid type, did -+-- Di hydroergotanhinc ---- krnoflov inc —1-- kIgoLiiuiilc • All not affect (p >0.10) CFU of either strain of E. co/i 0157:H7 in ruminal mixed cultures (data not IS shown). Regression analyses revealed only dihydroergotarnine tended (p=O.O8) to reduce CFU of E. co/i 0157:H7 strain 933 compared with individual ergot alkaloids or a combination of all 5 16 31.5 62.5 125 250 5110 1000 2 4 E. coli Concentration, uM three alkaloids (data not shown); CFU of 0157:H7 strain 6058 were not influenced FIG. 1. Maximal specific growth rates (MSGR per hour) (p> 0.10) by individual ergot alkaloids or a com- in pure culture of averaged Esclierichia coli 0157:H7 strain 933 after exposure to increasing concentrations of the bination of all three alkaloids (data not shown). ergot alkaloids dihydroergotam me, ergonovine, ergotamine, and a mixture of all three alkaloids at concentra- Discussion tions of 2 and 500 tM (ALl.). Growth of P. coP 0157:H7 was reduced ( p <0.003) when exposed to increased con- To our knowledge, these preliminary data are centrations of dihydroergotamine (250, 500, and 1000 pM) and ergotamine (1000 pM) hut not ergonovine. the first to test the influence of ergot alkaloids on growth of E. co/i 0157:H7. Individual alkaloids had mixed results on E. co/i 0157:H7; however, when dihydroergotamine, ergotamine, and er- combination of all three alkaloids compared to gonovine were combined, E. co/i 0157:H7 individual ergot alkaloids (Fig. 1). growth rate was decreased in both pure and mixed ruminal fluid cultures. These results Ruminal mixed culture suggest that mixtures of ergot alkaloids reduce Ergot alkaloids had no effect (p >0.10) on populations of F. co/i 0157:H7. Ruminants CFU for E. co/i 0157:H7 strain 933 after incu- consuming endophyte-infected tall fescue are bation in mixed ruminal fluid for 24 hours. exposed to a mixture of alkaloids (Bacon et a/., However, the mean number of CFU for F. coli 1977; Porter et a/., 1979; Lyons et a/., 1986). Ad- 0157:H7 strain 6058 was reduced (p <0.0001) ministration of both synthetic and natural ergot when exposed to a mixture of all three alkaloids peptide alkaloids is capable of mimicking most at any of the six concentrations (2, 8, 31.5, 125, of the symptoms and some physiological effects 500, or 1000 1.M) compared with individual al- of fescue toxicosis in animals (Browning et a/., kaloids (Table 2). Dihydroergotamine reduced 1998; Larson et al., 1999). Current results may (p =0.04) the number of E. co/i 0157:H7 strain explain our previous findings that fecal shed- 6058 CFU in ruminal mixed culture compared ding of E. coli 0157:H7 in naturally infected with ergonovine. The independent effects of cattle tended to be reduced in calves and was ergonovine and ergotamine resulted in a similar decreased in cows grazing endophyte-infected (p>O.lO) number of CFU of E. co/i 0157:H7 tall fescue compared with cows and calves (Table 2). The main effect of alkaloid concen- grazing endophyte-free tall fescue during the

TABLE 2. INFLUENCE OF THE MAIN EFFECTS OF ERGOT ALKALOIDS ON THE COLONY-FORMING UNITS (CFU) OF E. cOLI 0157;H7 STRAINS 933 AND 6058 IN MIXED RUMINAL CULTURE

Ergot alkaloid - CPU, Straw 933 CPU, Strain 6058

Dihydroergotamine 3.31 ± 0.07 3.74± 0.08h Ergonovine 3.54 10.07 3.99 0.08c Ergotamine 3.48 ± 0.07 3.84 ± 0.08bc Mixture of three alkaloids" 3.43 ± 0.09 3.04±0.11a

Values followed by the different letters are significantly different at p < 0.05. "Dihydroergotarnine, ergonovine, and ergotamine mixtures at each concentration (2, 8, 31.5, 125, 500, and 1000 1iM) independent of ergot alkaloid concentration.

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ERGOT ALKALOIDS AND GROWTH OF E. COLI 0157:H7 603

summer months (Looper et al., 2003). In con- steers receiving melatonin. Ergot alkaloids may trast, when ewes were experimentally inocu- influence E. co/i 0157:H7 growth either at the lated with E. co/i 0157:H7, fecal shedding cellular membrane level or via hormonal con- tended to be increased by consumption of en- trol, specifically, melatonin, or both. dophyte-infected tall fescue seed diets (Looper It is well established that cattle grazing endo- et al., 2007). Factors such as type of diet (grain- phyte-infected tall fescue experience a health based vs. grazing), different fecal shedding malady collectively characterized as fescue toxi- patterns between experimentally inoculated an- cosis which increases body temperature during imals and naturally infected animals, length of summer months, reduces reproductive perfor- exposure to the endophyte-infected tall fescue mance and growth rate, and decreases milk pro- and (or) reduced dry matter intake common in duction (Hoveland et at., 1983; Paterson et al., ruminants consuming endophyte-infected tall 1995). Results from the current experiment would fescue influence fecal shedding of pathogenic suggest that exposure to ergot alkaloids may re- bacteria (Callaway et al., 2003,2004; Looper etal., duce populations of E. co/i 0157:H7 in ruminants. 2007). Future studies utilizing naturally infected rumi- Dihydroergotamine reduced growth rate of nants are needed to determine the combination F. co/i 0157:H7 in pure and ruminal mixed of specific ergot alkaloids and the duration of cultures compared with ergonovine and ergot- exposure that reduce foodborne pathogens with- amine alone. Regression analyses suggest that out decreasing animal performance. E. co/i 0157:H7 growth is not reduced in a linear manner but only after a threshold concentration Acknowledgments of ergot alkaloid is reached. The specific mech- anisms by which the ergot alkaloids reduce Names are necessary to report factually on E. co/i 0157:H7 growth is unknown. Previous available data; however, the U.S. Department of research has suggested ergot alkaloids directly Agriculture does not guarantee or warrant the interact with membrane ATPase systems standard of the product, and the use of the name (Moubarak et al., 1993, 1998). It is well docu- by the U.S. Department of Agriculture implies mented that membrane Na and K-ATPase is no approval of the product to the exclusion of very important in maintaining membrane po- others that also may be suitable. tential and active transport of nutrients into cells (Skou and Esmann, 1992). An indirect effect of References E. co/i ergot alkaloids on 0157:H7 also may ex- Bacon CW, Porter JK, Robbins JD, and Luttrell ES. Epichioc ist. The structure of dihydroergotamine is sim- tiphina from toxic tall fescue grasses. AppI. Environ. ilar to serotonergic neurotransmitters and is a Microbiol. 1977;34:576-581. serotonin receptor agonist (Silberstein, 1997; Bouton j. The use of endophytic fungi for pasture im- Silberstein and McCrory, 2003). Synthesized by provement in the USA. Presented at the Grassland enterochromaffin cells from gastric and intesti- Conference, 4th International Neotyphodium/Grass in- teractions Symposium, Soest, Germany, 2000. nal mucosa, serotonin is the precursor to mela- Browning R Jr. Leite-Browning ML, Smith HM, and Wa- tonin which has been implicated in the kefield T. Effect of ergotamine and ergonovine on plas- seasonality of E. co/i 0157:H7 shedding in live- ma concentrations of thyroid hormones and cortisol in stock (Edrington et al., 2006). Fecal shedding is cattle. J. Anim. Sci. 1998;76:1644-1650. generally less in autumn and winter months Callaway TR, Anderson RC, Edrington TS, Genovese KJ, Bischoff KM, Poole TL, Jung YS, Harvey RB, and Nisbet (Hancock etal., 1997). A retrospective analysis of DJ. What are we doing about Eschcrichin co/i 0157:H7 in nine previously reported datasets revealed cattle? J . Anim. Sci. 2004;82(E. Suppl.):E93—E99. daylength was positively correlated with E. co/i Callaway TR, Elder RO, Keen JE, Anderson RC, and prevalence (Edrington et al., 2006). Steers graz- Nisbet DJ. Forage feeding to reduce pre-harvest E. coil ing endophyte-infected tall fescue had de- populations in cattle, a review. J . Dairy Sci. 2003;86:852- creased plasma concentrations of melatonin 860. Dunn JR, Keen JE, Del Vecchio R, Wittum TE, and et et al. (Porter al., 1993). Recently, Edrington Thompson RA. Eschericliia co/i 0157:H7 in a cohort of (2007) reported fecal shedding of E. coil 01 57:H7 weaned, preconditioned range beef calves. J . Food Prot. from naturally infected steers was decreased in 2004;67:2391-2396. 0

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Edrington TS, Callaway TR, Haliford DM, Chen L, An- Moubarak AS, Piper EL, and West CP. Effect of ergotamine derson RC, and Nisbet DJ. Effects of exogenous mela- and ergonovine on the renal ATPase system in the rat. tonin and tryptophan on fecal shedding of E. co/i Med. Sci, Res. 1993;21:289-290. 0157:H7 in cattle. Microb. Ecol. 2007;55:553-560. Moubarak AS, Rosenkrans CF Jr. and Johnson ZB. Effect of Edrington TS, Callaway TR, Ives SE, Engler MJ, Looper ergotamine and ergonovine on the bovine liver mito- ML, Anderson RC, and Nisbet DJ. 2006. Seasonal shed- chondrial ATPase system. Med. Sci. Res. 1998;26:699- ding of Esclierichia co/i 0157:H7 in ruminants: a new 701. hypothesis. Foodborne Pathog. Dis. 2006;3:413-421. Paterson J, Forcherio C, Larson B, Samford M, and Kerley Gansheroff U and OBrien AD. Escherichia co/i 0157:H7 in M. The effects of fescue toxicosis on beef cattle produc- beef cattle presented for slaughter in the U.S.: higher tivity. J. Anim. Sci. 1995;73:889-898. prevalence rates than previously estimated. Proc. Nat]. Porter JK, Bacon CW, and Robbins JD. Ergosine, ergosi- Acad. Sci. 2000;97:2959-2961. nine, and chanoclavine I from Epicli/oc tiphinn. J . Agric. Hancock DD, Besser TE, Rice DH, Herriott DL, and Tarr PT. Food Chem. 1979;27:595-598. A longitudinal study of Escherichia coli 0157 in fourteen Porter JK, Stuedemarin JA, Thompson FN, Buchanan cattle herds. Epidemiol. Infect. 1997;118:193-195. BA, and Tucker HA. Melatonin and pineal neurochem- Hoveland CS, Schmidt SP, King CC Jr. Odom JW, Clark icals in steers grazed on endophyte-infected tall fescue: EM, McGuire JA, Smith LA, Grimes HW, and Holliman effects of metoclopramidel. J . Anim. Sci. 1993;71:1526- JL. Steer performance and association of Acremonium 1531. coenophia!uni fungal endophyte on tall fescue pasture. Rangel JM, Sparling PH, Crowe C, Griffin PM, and Swer- 0157:H7 Agron. J. 1983;75:821-824. dlow DL. Epidemiology of Escheric/na co/i Laegreid WW, Elder RO, and Keen JE. Prevalence of Es- outbreaks, United States, 1982-2002. Emerg. Infect. Dis. cherichia co/i 0157:H7 in range beef calves at weaning. 2005;11:603-609. Epidemiol. Infect. 1999,-123:291-298. Shelby RA and Dalrymple LW. Incidence and distribution Larson BT, Harmon DL, Piper EL, Griffis LM, and Bush LP. of the tall fescue endophyte in the United States. Plant Alkaloid binding and activation of D2 dopamine re- Dis. 1987;71:783-786. ceptors in cell culture. J . Anim. Sci. 1999;77:942-947. Silberstein SD. The pharmacology of ergotarnine and di- Looper ML, Edrington TS, Flores R, Burke JM, Callaway hyd roergotamine. Headache 1997;37 (Suppl 1):15-25. TR, Aiken GE, Schrick FN, and Rosenkrans CF Jr. In- Silberstein SD and McCrory DC. Ergotamine and dihy- fluence of dietary endophyte-infected (NeoO,pliodi uin droergotamine: history, pharmacology, and efficacy. coenophialum) tall fescue (Fesfucn arimdinacea) seed on fe- Headache 2003;43:144-166. cal shedding of antibiotic-resistance selected Esclierichia Skou JC and Esmann M. The Na,K-ATPase. J. Bioenerg. co/i 0157:H7 in ewes. J . Anim. Sci. 2007;85:1102-1108. Biomembr. 1992;24:249-261. Looper ML, Edrington TS, Flores R, Rosenkrans CF Jr, Nihsen ME, and Aiken GE. Prevalence of Escl,ericlna co/i 0157:H7 and Salmonella in beef steers consum- Address reprint requests to: ing different forage diets. Lett. Appl. Microhiol. 20116;42: M.L. Looper, MS., Ph.D 583-588. Dale Bumpers Small Farms Research Center I .00per ML, Rosenkrans CF Jr. Aiken GE, and Edrington Agricultural Research Service TS. Escherichia co/i and Salmonella shedding in beef cattle grazing tall fescue. Ark. Agric. Exp. Stn. Res. Series 2003; U.S. Department of Agriculture 509:58-60. 6883 South State Hwy. 23 Lyons PC, Plattner RD, and Bacon CW. Occurrence of Booneville, AR 72927 peptide and clavine ergot alkaloids in tall fescue grass. Science 1986;232:487-489. E-mail: [email protected]