Research Note

Clostridium perfringens Alpha 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 Science, and †Clinic for Avian, , and Medicine, University of Veterinary Medicine, Veterina¨rplatz 1, A-1210 Vienna, Austria

ABSTRACT that colonize the intestinal tract to the serosal side induced a biphasic increase in short- can invade epithelial cells or produce that cause circuit current (Isc) after 15 and 100 min. The magnitude diarrhoeal diseases. Proliferation of 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 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 . 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 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 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. Later, the Ringer solution intestinal contents several times with Ringer solution at in the mucosal side was replaced by the same solution 4°C. Tissues were placed in the same cold Ringer buffer with 5 mmol/L of D-glucose instead of mannitol. The and gassed with carbogen (O2/CO2, ratio 95:5) until they electrical response was measured as the peak response were mounted in the Ussing chamber. obtained 1 min after the addition of glucose. Six jejunal 1300 REHMAN ET AL.

Table 1. Short-circuit current (␮A/cm2) of the isolated jejunal tissues Table 2. Transmural potential difference (mV) across the isolated jejunal from laying hens with or without preincubation with alpha toxin1 tissues from laying hens with or without preincubation with alpha toxin1

Toxin Toxin Time Control preincubated P-value Time Control preincubated P-value

Basal 42 ± 28 31 ± 11 0.62 Basal −3 ± 2 −2 ± 1 0.51 After glucose addition 103 ± 27 44 ± 13 0.03 After glucose addition −7 ± 2 −3 ± 1 0.059 30 minutes post glucose addition 23 ± 30 16 ± 12 0.80 30 min post glucose addition −2 ± 2 −2 ± 1 0.78

1Values are expressed as means ± SEM of 7 birds (3 replicates/bird). 1Values are expressed as means ± SEM of 7 birds (3 replicates/bird).

Experiment 2 strips were prepared from individual birds in each set of experiment. Addition of D-glucose to the mucosal compartment re- sulted in an increase of Isc in treated and control tissues Statistics (Table 1) when compared with their respective basal val- ues. In control tissues that were not preincubated with ␮ 2 Statistical program SPSS (version 11.5, SPSS GmbH, toxin, the increase of Isc (∆Isc) was 60.6 A/cm , whereas SPSS Inc., Munich, Germany) was used for data analysis. the tissues incubated with alpha toxin had a diminished The Kolmogorov Smirnov test was used to test the normal response of 13.4 ␮A/cm2 compared with the baseline < distribution of the data. Results are expressed as means Isc (P 0.05). The addition of D-glucose decreased the ± SEM. Repeated measures ANOVA procedure was used transmural potential difference in the control group as to investigate the effect of alpha- and D-glucose on the Isc, well as in the jejunal sheets incubated with toxin; there tissue conductance, and transmural potential difference (3 was a greater decrease (P = 0.059) in the control group replicates/bird) and subsequent paired t-test. In Experi- compared with the treatment group (Table 2). The tissue ment 2, independent sample t-test was used for compari- conductivity was not affected at any time interval when son between control and toxin group. Probability values control and toxin-treated groups were compared (Table of less than 0.05 (P < 0.05) were considered significant. 3).

RESULTS DISCUSSION In chickens, the toxin of C. perfringens has been impli- Experiment 1 cated in the pathogenesis of NE with diarrhoea and mac- roscopic lesions in the small intestine. The incidence of After an equilibration time of 30 min, the tissues were NE in poultry has increased in many countries, probably short-circuited until a stable basal line was achieved prior due to the reduced utilization of antibiotic growth pro- to the addition of alpha toxin. No change in the Isc was moters (Immerseel et al., 2004). observed when the toxin was added to the mucosal com- To our knowledge, no study has been carried out to partment of the Ussing chamber. Even the addition of a investigate in vitro the effect of alpha toxin of C. per- 3 higher concentration of alpha toxin (10 U/L) failed to fringens on the sodium, chloride, or glucose transport in elicit any change in Isc when added to the mucosal side chicken intestine, and only scanty data are available in (data not shown). However, administration of alpha toxin other species (Diener et al., 1991; Miyakawa and Uzal, in a concentration of 100 U/L to the serosal side induced 2005). The experiment described in the present report a biphasic increase in Isc in the jejunal strips. The first demonstrated an effect of purified alpha toxin on the 2 increase (44 ± 13 ␮A/cm ) was observed after 15 min, electrophysiology of the intestine of laying hens. Addition < ± which was higher (P 0.001) than the basal Isc (22 12 of alpha toxin to the luminal compartment of the Ussing ␮ 2 A/cm ). Afterwards, the Isc began to fall and approached chamber did not induce changes in the Isc. The results to the baseline (P > 0.05) within 40 min (10 ± 10 ␮A/cm2). are in agreement with studies in rats (Diener et al., 1991), Then, it started to rise again in the second phase, and which also failed to observe changes in the Isc when alpha ± maximum response of alpha toxin in terms of Isc (41 11 toxin was added to the mucosal side of rat colon. It may ␮A/cm2) was observed after 100 min which was higher (P < 0.05) than the base-line (22 ± 12 ␮A/cm2). The second 2 phase of Isc response was statistically not different in Table 3. Tissue conductance (mS/cm ) of the isolated jejunal tissues 1 magnitude from the first phase (P > 0.05); however, the from laying hens with or without preincubation with alpha toxin response of the second phase remained for a longer time, Toxin and it reached the basal value after 200 min (−5 ±13 ␮A/ Time Control preincubated P-value 2 > 2 cm , P 0.05). The tissue conductance (mS/cm ) tended Basal 13 ± 219± 3 0.25 to be lower (P = 0.07) 2 h postaddition of alpha toxin (9 After glucose addition 12 ± 116± 2 0.29 ± ± ± 5) compared with the basal value before the administra- 30 min post glucose addition 14 6192 0.29 tion of alpha toxin (11 ± 5). 1Values are expressed as means ± SEM of 7 birds (3 replicates/bird). RESEARCH NOTE 1301 be speculated that additional co-factor is required for this the net flux of sodium from the luminal to the serosal toxin to elicit its action on mucosal membrane of the side. All of these events modify the electrical parameters gastrointestinal tract. This toxin may bind with mucus, of the intestinal tissue and increase Isc (Shimada and thus hampering its diffusion to the apical membrane of Hoshi, 1986; Wright et al., 1994; Amat et al., 1999). In the the enterocytes. Clostridium perfringens is also able to grow present study, the addition of 5 mmol/L of D-glucose to on medium with mucin as a substrate, indicating that the mucosal side caused a significant increase in Isc in this organism possesses mucolytic activity (Deplancke et control tissues and the tissues preincubated with alpha al., 2002). The sequence of C. perfringens showed toxin (Table 1), which is due to the stimulation of transepi- that this bacterium also secretes exo-alpha sialidase thelial Na+ flux. The response to mucosal addition in (Shimizu et al., 2002). Mucolysis could serve as an initiat- toxin-exposed jejunum was decreased (P < 0.05) in com- ing step of C. perfringens virulence and point to a possible parison with the response of the control tissue. The de- link between bacterial mucolysis in the chick’s intestine creased response to glucose could indicate that diarrhoea and intestinal barrier function (Collier et al., 2003). caused by the alpha toxin is not only secretory and chlo- Alpha toxin induced a biphasic increase in Isc when the ride mediated but also caused by sodium malabsorption toxin was added to the serosal compartment of the Ussing or, at least, by a substantially diminished substrate-de- chamber. The first peak was attained after 15 min, and pendent Na+-absorption from the jejunum. This toxin is the second peak was reached after about 100 min. The also involved in excess secretion of mucus (Miyakawa results are similar to the findings of Diener et al. (1991) and Uzal, 2005). The toxin may increase the production who also observed 2 peaks when rat colon was exposed of mucus that might have caused an increase in the thick- to the alpha toxin on the serosal surface. The second peak, ness of the unstirred layer surrounding the enterocytes, in the present study, did not differ from the first peak (P thus impeding the diffusion of glucose toward the apical > 0.05) in terms of magnitude of Isc; however, Diener et membrane of the enterocytes. al. (1991) demonstrated a higher increase in ∆Isc in the The tissue conductance was decreased (P = 0.07) 2 h second peak as compared with the first peak. It is possible postaddition of alpha toxin. This may be due to an in- that alpha toxin from different isolates of C. perfringens crease in paracellular or transcellular resistance or both. toxotype or even from different isolates of C. perfringens On the other hand, Otamiri (1989) suggested that this type A have different morphologic or physiological ef- toxin might impair the function of the mucosal barrier fects on the intestine (Miyakawa and Uzal., 2005). and increase the permeability of the intestine. Therefore, The ionic mechanism of the increase in Isc in the first this aspect needs further experimentation involving the peak may be due to the electrogenic secretion of chloride role of various proteins of the tight junction of enterocytes anions. The first and immediate response may be due to in the mediation of regulation of barrier function of the the release of arachidonic acid, probably from the connec- gastrointestinal tract following alpha toxin treatment. tive tissue of the submucosa and lamina propria. The In conclusion, the serosal addition of alpha toxin caused toxin first encounters the connective tissue of submucosa a biphasic increase in Isc in jejunum sheets from laying and lamina propria before penetrating to the enterocytes. hens. Both peaks were similar in magnitude, but the sec- The second phase of alpha toxin response may be due to ond peak remained for a longer time. 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