Research Note Clostridium perfringens Alpha Toxin Affects Electrophysiological Properties of Isolated Jejunal Mucosa of Laying Hens H. Rehman,* W. A. Awad,* I. Lindner,† M. Hess,† and J. Zentek*1,2 *Institute of Nutrition, Department of Veterinary Public Health and Food Science, and †Clinic for Avian, Reptile, and Fish Medicine, University of Veterinary Medicine, Veterina¨rplatz 1, A-1210 Vienna, Austria ABSTRACT Bacteria that colonize the intestinal tract to the serosal side induced a biphasic increase in short- can invade epithelial cells or produce toxins that cause circuit current (Isc) after 15 and 100 min. The magnitude diarrhoeal diseases. Proliferation of Clostridium per- of the increase of Isc of both peaks was similar, but the fringens and production of alpha-toxin, a phospholipase second phase response lasted longer. The tissue conduc- C, is the major factor for necrotic enteritis in poultry. tivity tended (P = 0.07) to be lower after 2 h of toxin However, little is known about the functional importance addition compared with basal value when no toxin was of luminal alpha-toxin during intestinal infection. The added. In the second experiment, adding D-glucose on purpose of this study was to investigate the effects of the mucosal side of the jejunum increased (P < 0.05) the ± ␮ 2 purified alpha toxin of Clostridium perfringens on the elec- Isc from a baseline value of 42 28 A/cm to a maximal trophysiology of the laying hen’s stripped jejunum in value of 103 ± 27 ␮A/cm2. Preincubation with α-toxin Ussing chambers. The effects were investigated in Experi- almost fully inhibited this stimulation of Isc by D-glucose. ment 1 after toxin addition to the mucosal and serosal The conductance of the tissues was not affected by the side of the tissue, and a second experiment was performed toxin addition. These findings indicate that alpha toxin to study the effect of the toxin on sodium-dependent not only causes electrogenic secretion of anions, probably glucose transport. Mucosal exposure of jejunal tissue due to the stimulation of chloride secretion, but also di- sheets to 100 units of alpha toxin/L did not elicit electro- minishes electrogenic Na+/glucose cotransport from the physiologic changes. The addition of purified alpha toxin mucosal to serosal side in the small intestine of poultry. Key words: laying hen, Clostridium perfringens, alpha toxin, phospholipase C, Ussing chamber 2006 Poultry Science 85:1298–1302 INTRODUCTION (Titball et al., 2000). Phospholipases are a group of en- zymes, and the role of these enzymes in the pathogenesis Clostridium perfringens, a widely distributed pathogen, of infectious disease is diverse. A key event is the interac- is a low G + C gram-positive anaerobic spore-forming tion of the enzyme with phospholipids in eukaryotic cell bacterium (Shimizu et al., 2002). This bacterium is ubiqui- membranes (Titball, 1993). Alpha toxin exhibits haemo- tous in the environment and a member of the normal gut lytic, necrotic, vascular permeabilizing, and platelet ag- flora of animals (Collier et al., 2003). Clostridium per- gregating properties (Titball, 1993; Titball et al., 1999). It fringens produces different toxins (Smedley et al., 2004), hydrolyzes the phosphatidylcholine and sphingomyelin and on the basis of the ability to produce major lethal moieties in the presence of calcium ions and promotes toxins, the alpha, beta, epsilon, and iota, C. perfringens membrane disorganization (Titball, 1993; Naylor et al., isolates are grouped into 5 toxin types (Type A–E; Mac- 1998; Titball et al., 1999). Hydrolysis of lecithin results in Lennan, 1962; McDonel, 1980). The alpha toxin from C. a formation of diacylglycerol, resulting in the activation perfringens was the first bacterial protein shown to possess of protein kinase C and subsequent stimulation of the enzymatic and toxic properties (MacFarlane and Knight, arachidonic acid cascade. This induces the synthesis of 1941). This toxin has a phospholipase C (PLC) activity inflammatory mediators such as leukotriens, thrombox- and a calcium-dependent phospholipid binding domain ans, platelet-agglutinating factors, and prostacyclins (Tit- ball, 1993; Bunting et al., 1997; Titball et al., 1999). Analysis of the alpha toxin of chicken isolates of C. perfringens 2006 Poultry Science Association Inc. demonstrated high homology to mammal-derived strains Received January 5, 2006. (Sheedy et al., 2004). Conditions that promote the exces- Accepted February 25, 2006. sive growth of C. perfringens in chicken intestine lead to 1 Corresponding author: [email protected] toxin production that causes necrotic enteritis (NE). 2Present address: Institute of Animal Nutrition, Faculty of Veterinary Medicine, Free University of Berlin, Bru¨ mmerstr. 34, D-14195 Berlin, When alpha toxin was inoculated into ligated intestinal Germany. loops of adult pigs, it did not induce substantial lesions 1298 RESEARCH NOTE 1299 or fluid loss (Jestin et al., 1985). However, when adminis- Chemicals tered intragastrically to neonatal piglets, alpha toxin causes edema of villi and neutrophilic inflammation of α-Toxin [phospholipase C (Sigma-Aldrich, Deisenho- the small intestine (Johannsen et al., 1993a,b). Administra- fen, Germany), EC 3.1.4.3, Type 1] from C. perfringens was tion of semipurified ultrafiltered alpha toxin in ovine ileal dissolved in Ringer solution. The specific activity of PLC and colonic loops in vivo induced accumulation of fluid was 25 units/2.6 mg of solid. In each experiment, alpha in the lumen (Miyakawa and Uzal, 2005). Alpha toxin toxin was reconstituted freshly. All the salts used to pre- induced the contraction of isolated ileum (Sakurai et al., pare the Ringer solution were of analytical grade and 1990) and neutrophilic enteritis when injected into small were purchased from Sigma-Aldrich. intestinal loops of rats (Otamiri, 1989). Serosal addition of 100 units of purified PLC to rat colon has been reported Measurements of to induce a biphasic current because of chloride ion de- Electrophysiological Traits pendent secretion (Diener et al., 1991). In poultry, the functional importance of intestinal alpha Short-circuit current (Isc), tissue conductance, and toxin release in the pathogenesis of NE is unclear. To transmural potential difference were measured in the our knowledge, no information is available regarding the Ussing chamber with a microprocessor system based on changes in the electrophysiological parameters of the a voltage/current clamp device (Mussler, Microclamp, small intestine and glucose absorption induced by alpha Aachen, Germany). toxin of C. perfringens. Therefore, the electrophysiological The serosa and muscularis were stripped manually to changes and the effects on electrogenic sodium glucose obtain a mucosa-submucosa preparation of the jejunum. transport were studied in vitro in jejunal strips from lay- Thereafter, the epithelial sheets were mounted in the ing hens in the Ussing chambers. modified Ussing chambers with an exposed tissue area of 1 cm2. The serosal and mucosal surfaces of the tissues MATERIALS AND METHODS were bathed in 5 mL of Ringer solution with the same composition described previously. The bathing medium Birds, Feeding, and Housing in the chambers was aerated with 95% O2 and 5% CO2 and maintained at 38°C in a water bath (Julabo Inc., Allen- Lohmann Brown laying hens (n = 13), 72 wk of age, town, PA). The solution was continually stirred and oxy- were procured from a local commercial farm (O¨ sterr. Sup- genated by bubbling into the chamber by means of a gas penhu¨ hner Verwertungs AG, Weistrach, Austria). The lift. The electrode potential and the solution resistance hens, weighing 1.5 to 2.0 kg, were housed on deep wood were determined at the beginning of every experiment shavings litter and were fed a commercial diet (Tagger and were automatically corrected before tissues were Feed Mill, Graz, Austria). The diet contained 17.0% crude placed in the chamber. The tissues were first incubated protein, 5.0% crude fat, 4.3% crude fiber, 13.3% crude ash, under open circuit conditions for 30 min for equilibration and 0.4% methionine. The hens were provided diets and and were short-circuited after that by clamping the volt- water ad libitum for the duration of the experiment. The age at 0 mV. The toxin was added when the base line actual experiment was started a week after the arrival of was achieved. the birds in order to acclimatize them in a new envi- ronment. Experimental Design Tissue Preparation Experiment 1. This experiment was performed to de- termine whether alpha toxin stimulates ionic secretion The hens were killed by stunning and bleeding at the (n = 6). The toxin was added to the mucosal compartment Institute of Nutrition, University of Veterinary Medicine, of the Ussing chamber where no response of the toxin Vienna, Austria. The jejunum was then removed directly was noted on the short-circuit current. Further experi- after exsanguination, rinsed several times with ice-cold ments were performed to investigate the effects of toxin Ringer buffer (4°C), and transported in ice-cold oxygen- after addition to the serosal compartment of the Ussing ated buffer to the laboratory within 5 min. The composi- chamber. The final concentration of the toxin was 100 U/ tion (mmol/L) of Ringer solution was CaCl2, 1.2; MgCl2, L of Ringer solution. 1.2; Na2HPO4, 2.4; NaH2PO4, 0.4; NaHCO3, 25; KCl, 5; Experiment 2. The second experiment was carried out NaCl, 115; and mannitol, 20; the pH was adjusted to 7.4. to investigate the effect of alpha toxin on sodium-depen- The segments taken from the proximal jejunum and tissue dent glucose absorption (n = 7). The proximal jejunum flaps were prepared by the method as described by Awad sheets were preincubated with the toxin (100 U/L) for et al. (2005b). The intestinal segment was opened longitu- 30 min. Thereafter, tissues were short-circuited unless a dinally along the mesenteric border and washed free of stable base line was achieved.