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Poult Sci 2007. 86:2358-2366. doi:10.3382/ps.2007-00133
© 2007 Poultry Science Association
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METABOLISM AND NUTRITION

Effect of Different Dietary Methionine Sources on Intestinal Microbial Populations in Broiler Chickens

J. P. Dahiya*, D. Hoehler{dagger}, A. G. Van Kessel* and M. D. Drew*,1

* Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5A8; and {dagger} Degussa Corporation, Kennesaw, GA 30144

1 Corresponding author: murray.drew{at}usask.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two experiments were conducted to study the effect of various levels of DL-Met or 2-hydroxy-4-methylthiobutanoic acid (MHA-FA) on Clostridium perfringens and other intestinal bacteria in broiler chickens. In each experiment, 2 cages of 6 birds (14 d posthatch) were assigned to 1 of 7 different diets in a 2 x 4 factorial arrangement. The main effects were Met source (either DL-Met or MHA-FA) and Met level (0, 0.2, 0.4, or 0.8% DL-Met or 0, 0.227, 0.454, and 0.908% MHA-FA, thus providing 4 corresponding equimolar levels of each Met source). All birds were orally gavaged with a C. perfringens type A broth culture on d 1 and on d 14 to 20 and killed on d 28. Intestinal populations of C. perfringens, lactobacilli, Streptococcus group D, and coliforms were enumerated in the ileum and cecum, and necrotic enteritis intestinal lesions were scored. There was a significant reduction (P < 0.05) in C. perfringens populations in birds fed either Met source in the cecum (experiment 1) or the ileum and cecum (experiment 2). In experiment 2, the lactobacillus populations were significantly higher (P < 0.05) in the ceca of birds receiving 0.8% Met than in the birds given diets with the other levels of Met tested. Significantly lower populations (P < 0.05) of coliforms and Streptococcus group D were enumerated in the ileum of birds fed the 0.8% Met-supplemented diet than in the other dietary treatments. The effect of Met source on intestinal bacteria was not significant, suggesting that both DL-Met and MHA-FA have similar antibacterial properties. Last, there were no significant differences in intestinal lesion scores or the performance of birds fed different Met sources and concentrations. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of C. perfringens in broiler chickens when used in relatively high concentrations, and may reduce the risk of necrotic enteritis. Thus, feeding low-protein diets supplemented with crystalline amino acids might be beneficial in terms of the growth of various enteric pathogens.

Key Words: broiler • DL-methionine • 2-hydroxy-4-methylthiobutanoic acid • necrotic enteritis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clostridial or necrotic enteritis (NE) caused by Clostridium perfringens type A and C (Ficken and Wages, 1997) is a potentially fatal poultry disease of global significance, and is both an animal welfare and economic problem. Outbreaks of NE are sporadic and may result in high mortality, reduced growth rate, impaired feed conversion, and increased condemnation rates (Lovland and Kaldhusdal, 2001). Necrotic enteritis is estimated to cost the poultry industry as much as US $0.05 per bird, with a total global loss of nearly US $2 billion (Van der Sluis, 2000). However, this might be an underestimate, given the difficulty in diagnosing mild or subclinical forms of NE. In recent years, the banning of prophylactic use of antibiotics in Europe, as well as consumer concerns about their use in other jurisdictions, has stimulated interest in finding alternative management or dietary strategies to control the incidence and severity of this disease in the postantibiotic era.

Clostridium perfringens is frequently found in the intestinal tract of healthy poultry, usually at low levels (< 104 cfu/g of digesta) and is spread in poultry production units and processing plants through feces and intestinal rupture. Clostridium perfringens is the main etiological agent of NE, although other cofactors are usually required to precipitate an outbreak of NE (Dahiya et al., 2006). The physical and chemical composition of broiler diets has been reported to have a marked effect on the intestinal microflora of chickens, and it has been shown to have an important impact on the incidence of NE in broiler chickens (Riddell and Kong, 1992; Drew et al., 2004; Dahiya et al., 2006). Dietary cereal grains rich in nonstarch polysaccharides and dietary proteins, especially proteins of animal origin, encourage the development of NE (Riddell and Kong, 1992; Kaldhusdal and Skjerve, 1996; Annett et al., 2002; Wilkie et al., 2005).

Drew et al. (2004) observed a significant increase in C. perfringens counts in birds fed a 40% CP fish meal diet; however, C. perfringens counts were low in the ileum of birds fed soy protein concentrate-based diets at all levels of CP. A significant positive correlation between the Gly content of the diets and digesta and C. perfringens populations in the ileum and cecum of broiler chickens has been reported (Dahiya et al., 2005, 2007; Wilkie et al., 2005). Some in vitro studies have shown an association between certain amino acids and C. perfringens growth, {alpha} toxin production, or both (Ispolatovskaya, 1971; Muhammed et al., 1975; Nakamura et al., 1978; Titball et al., 1999; Stevens and Rood, 2000). Ispolatovskaya (1971) and Stevens and Rood (2000) reported that Gly or Glycontaining peptides accelerated C. perfringens growth and {alpha} toxin production in vitro. Although Muhammed et al. (1975) documented that Met was stimulatory for the growth of C. perfringens in vitro, previous experiments in our laboratory have demonstrated an antibacterial effect of high concentrations of DL-Met against C. perfringens in in vitro experiments (Wilkie et al., 2005; Wilkie, 2006). These researchers reported a significantly reduced growth of C. perfringens after a 24-h incubation of mixed bacterial culture in minimal salt media supplemented with a 10 mg/mL solution of DL-Met, compared with unsupplemented media.

Methionine is commonly supplemented as dry DL-Met (99% pure) or as liquid DL-Met hydroxy analog-free acid at concentrations ranging from 0.10 to 0.25% in poultry diets. Based on our preliminary results, we hypothesized that high dietary concentrations of Met are responsible for reduced C. perfringens growth in the gastrointestinal tract of broiler chickens and thus may decrease the risk of NE. Hence, the purpose of this experiment was to determine the effect of nutritional and supranutritional levels of 2 Met sources [DL-Met and 2-hydroxy-4-methyl-thiobutanoic acid (MHA-FA)] on intestinal C. perfringens and other microbial populations, and on NE lesion scores in broiler chickens.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental protocols were approved by the Animal Care Committee of the University of Saskatchewan and were performed in accordance with recommendations of the Canadian Council on Animal Care as specified in the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, 1993).

Experimental Animals, Diets, and Design

In each experiment, a total of 84 one-day-old conventional male broiler chicks (Ross 308) were obtained from a local broiler hatchery (Lilydale Hatchery, Wynyard, Saskatchewan, Canada) and housed randomly in 7 electrically heated battery cages for the first 2 wk of age (12 birds per cage) at the Animal Care Unit, Western College of Veterinary Medicine of the University of Saskatchewan. The birds received a medicated, ideal protein-balanced (3,200 kcal/kg of ME; 1.2% Lys) corn-based starter crumble (Coop Feeds, Saskatoon, Saskatchewan, Canada) for the first 14 d of the experiment (Table 1Go). On d 14, the birds were weighed and 2 cages of 6 birds each were assigned in a completely randomized design to 1 of the 7 different ideal protein-balanced experimental diets in a 2 x 4 factorial arrangement. The main effects were Met source (DL-Met or MHA-FA) and Met level (0, 0.2, 0.4, and 0.8% DL-Met or 0.227, 0.454, and 0.908% MHA-FA, thus providing 4 corresponding equimolar levels of each Met source; Tables 2Go and 3Go). The control diet was formulated to contain 23% CP, 1.2% Lys, 0.38% Met, and 3,200 kcal/kg of ME. The control diet was then supplemented with either dry DL-Met or liquid MHA-FA to achieve 4 equimolar levels of each Met source (0, 0.2, 0.4, and 0.8%). The diets met or exceeded the NRC (1994) energy and nutrient requirements for broiler chickens for all other nutrients.


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  		Table 1. Composition of starter diet1 used in both experiments up to d 14 of age
 

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  		Table 2. Experimental design
 

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  		Table 3. Composition of experimental diets used from d 14 to 28 of age in experiments 1 and 2 (% as-is basis)
 
Standard management procedures were followed during this study for both experiments. The research facility was thoroughly cleaned and disinfected prior to bird placement. The battery cages were arranged in 4 tiers with wire floors and were equipped with external feed and water troughs. The lighting schedule was 16L:8D throughout the experiment, with controlled temperature and humidity. Room temperature was maintained according to industry standards. None of the experimental diets contained antibiotics or coccidiostats, and diets were not pelleted. Throughout the experimental period in each experiment, birds had ad libitum access to food and water. Feed consumption and BW for each cage were recorded for the period of d 14 to 21 and d 21 to 28 in each experiment to calculate feed conversion. When calculating feed conversion, the BW of dead birds was taken into account. Amino acid analysis of the different diets was performed by the Degussa Corporation (Degussa Canada Inc., Burlington, Ontario, Canada).

C. perfringens Challenge Model

The C. perfringens challenge model was based on the model originally developed by Dahiya et al. (2005), with some modifications. Briefly, an avian C. perfringens field strain isolated from a clinical case of NE was obtained from Manuel Chirino, College of Veterinary Medicine, University of Saskatchewan, and characterized by the PCR technique as a type A toxin producer. The organism was cultured anaerobically on BBL blood agar base (Becton, Dickinson and Co., Sparks, MD) containing 5.0% sheep blood and 100 mg/L of neomycin sulfate (The Upjohn Company) for 18 h at 37°C, and then aseptically inoculated into either brain heart infusion (Difco Labs, Detroit, MI; experiment 1) or cooked meat medium (Difco Labs; experiment 2) and incubated anaerobically overnight (experiment 1) or for 8 h (experiment 2) at 37°C. All birds were orally challenged in the crop with this actively growing culture of C. perfringens with 0.5 mL on d 1 and 1.0 mL on d 14 to 20, inclusive, by using a 12.0 mL syringe equipped with vinyl tubing (i.d. 0.97 mm, o.d. 1.27 mm). Bacterial counts were performed on the culture daily prior to inoculation, and the numbers ranged from 4.51 x 104 to 3.73 x 106 cfu/mL.

Pathological Examination

Birds were observed on a pen basis at least once daily for any signs of NE (e.g., huddling, diarrhea, depression, or mortality), and all birds that died during the course of the experiments were necropsied to determine the cause of death. On d 28, the surviving chickens were killed by cervical dislocation, weighed, and necropsied immediately. Intestinal tracts were removed and intestinal lesions were scored blindly according to the method of Truscott and Al-Sheikhly (1977), with slight modifications, on a scale of 0 to 4 as described previously by Dahiya et al. (2005). Following postmortem examination, if the score was ≥ 1 then a 1.5- to 2.0-cm-long piece of intestinal tissue with the gross lesion was collected in phosphate-buffered formaldehyde solution and processed routinely for paraffin embedding, sectioned at approximately 5 µm, and stained with hematoxylin and eosin.

Bacterial Enumeration

As described above, on d 28, birds were selected at random from each pen, weighed, and killed by cervical dislocation and their intestinal tracts were removed. Samples of fresh digesta (0.1 to 0.2 g) from the ileum (Meckel’s diverticulum to 1 cm proximal to the ileocecal junction) and ceca were collected aseptically in preweighed 15-mL sterilized plastic tubes containing 1 mL of 0.1% sterile peptone buffer with 5 g/L of Cys hydrochloride (Sigma Chemical Co., St. Louis, MO). The digesta samples were pooled from 2 birds from each cage. The samples were immediately placed on ice and kept there until plated, within 3 h of collection. The samples were weighed and diluted in peptone water to an initial 10–1 dilution. Tenfold dilutions were spread in duplicate with an automated spiral plater (Autoplate, Spiral Biotech Inc., Bethesda, MD) on BBL blood agar base (Becton, Dickinson and Co.) containing 5% sheep blood and 100 mg/L of neomycin sulfate (The Upjohn Company) for C. perfringens enumerations. In addition, all the digesta samples were cultured on de Man, Rogosa, Sharpe agar (Becton, Dickinson and Co.), MacConkey’s agar (Becton, Dickinson and Co.), and bile esculin agar (Becton, Dickinson and Co.) for enumeration of lactobacilli, coliforms, and Streptococci group D, respectively. The plates were incubated at 37°C for 16 to 24 h anaerobically for C. perfringens and lactobacilli and aerobically for coliforms and Streptococcus group D bacteria. The {alpha}- and β-hemolytic colonies on blood agar-neomycin plates were counted as C. perfringens, with presumptive colonies being randomly picked, gram stained, plated on mannitol yolk polymixin agar (Oxoid Inc., Nepean, Ontario, Canada), and examined microscopically to confirm them as C. perfringens. Counts were expressed as log10 cfu per gram of intestinal contents.

Statistical Analysis

The data were analyzed by the GLM procedure of SPSS (v. 12.0, SPSS Inc., Chicago IL) as a 2 x 4 factorial (2 Met sources, each with 4 levels). All the effects were considered as fixed, and the interactions between Met source and level were used in the model, with pen as the experimental unit. There was no pen effect on any of the measured parameters in either experiment. The digesta samples for bacterial enumeration were pooled from 2 birds from each pen, resulting in 6 observations for each diet in each experiment. The treatment means were compared by using the Ryan-Einot-Gabriel-Welch multiple F-test, and comparisons were deemed significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The CP and amino acid composition of the experimental diets used in this study from d 14 to 28 of age is shown in Table 3Go. The dietary CP, DL-Met, and MHA-FA concentrations were very close to our planned levels. All of the other amino acids and MHA-FA were in similar concentrations in all experimental diets in both experiments.

In both experiments, some of the birds initially became dull and depressed, and had abnormally wet droppings after the C. perfringens challenge was initiated on d 14. During the course of this study, 2 birds died in experiment 1 (1 each from the control and 0.4% DL-Met-supplemented groups) and 3 birds died in experiment 2 (1 each from the control, 0.2, and 0.8% MHA-FA-supplemented groups) of unknown causes. The majority of dead birds were in good condition and had no typical field-type gross lesions of NE in either the intestine or any other organ that might have caused the death. Only 1 bird had a distended jejunum and ileum with a thin and friable intestinal wall, and the lumen was filled with gas and dark brown fluid content. There were few petechial hemorrhages in the distal jejunum and proximal ileum in the remaining dead birds, whereas the surviving birds displayed no obvious signs of morbidity at 7 to 10 d postchallenge.

The average feed consumption, BW gain, and feed conversion for the periods of d 14 to 21 and d 21 to 28 of age were not significantly different among various dietary treatments in any of the experiments (data not shown). In addition, there was no significant interaction between Met source and level for various performance parameters.

On d 28 of age, the populations of all 4 bacterial species enumerated in this study (C. perfringens, lactobacilli, Streptococcus group D, and coliforms) were higher in the cecum than in the ileum of broiler chickens. There were no significant differences in the growth of various bacterial species in the intestinal tract of broiler chickens fed 2 different Met sources, whereas Met concentration had a significant effect on various bacterial species either in the ileum or cecum, or both (Tables 4Go and 5Go). There was no significant interaction between Met source and Met concentration for various bacterial populations in either the ileum or cecum in either experiment. The only exception was C. perfringens growth in the ileum and cecum in experiment 2, where there was a significant interaction between Met source and level. There was a decrease in the numbers of C. perfringens with an increase in dietary DL-Met in both the ileum and cecum, whereas C. perfringens counts were higher in the ileum (3.38 vs. 3.90 log10 cfu/g of wet digesta) and cecum (3.98 vs. 4.98 log10 cfu/g of wet digesta) when dietary MHA-FA was increased from 0.2 to 0.4%. However, there were no significant differences in C. perfringens counts between DL-Met and MHA-FA at the 0.2 or 0.4% inclusion rates.


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  		Table 4. Effect of different Met sources or levels on various bacterial populations in the ileum of broiler chickens on d 28 of age in experiments 1 and 21
 

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  		Table 5. Effect of different Met sources or levels on various bacterial populations in the ceca of broiler chickens on d 28 of age in experiments 1 and 21
 
In experiment 2, significantly higher numbers of C. perfringens were observed in the ileum of birds fed the control diet as compared with birds given Met-supplemented diets. In addition, C. perfringens populations in the ileum decreased significantly with an increase in Met supplementation from 0.2 to 0.8%. In experiment 1, C. perfringens growth in the ileum was not significantly different among various dietary treatments. In experiment 1, the ceca of birds fed either 0.2 or 0.4% Met contained significantly lower C. perfringens counts than the ceca of birds in the control diet with no supplemental Met. The control diet supported a significantly higher growth of C. perfringens than the Met-supplemented diets in experiment 2. An important point to note is that the C. perfringens populations were generally higher in both the ileum and cecum in experiment 2 compared with experiment 1.

The lactobacillus populations were not significantly altered in the ileum of broiler chickens fed different levels or sources of Met in the 2 experiments (Table 4Go). Similarly, there were no significant differences in lactobacilli growth in the cecum in experiment 1. However, in experiment 2, the lactobacillus populations were significantly higher in the ceca of birds receiving 0.8% Met than in those of birds given diets with the other levels of Met tested (Table 5Go).

In experiment 2, Streptococcus group D populations were significantly lower in the ileum of birds receiving 0.8% Met-supplemented diets compared with those given the control, 0.2, or 0.4% Met-supplemented diets. We observed a significant difference in Streptococcus group D growth in the ceca of birds receiving the control diet and the 0.2% Met-supplemented diet in experiment 1, whereas there were no significant differences in the ceca of birds in experiment 2.

In experiment 2, the coliform counts were significantly higher in the ileum of broiler chickens that were given the control diets with no added Met compared with birds receiving the rest of the experimental diets with different concentrations of Met. Methionine source or level had no significant impact on coliform counts in the cecum in either experiment 1 or 2 (Table 5Go). The coliform counts were substantially lower than those of lactobacilli and streptococci in the intestines of chickens on d 28 of age.

The mean NE lesion score of chickens fed different experimental diets and killed on d 28 is depicted in Figure 1Go. Dietary Met source or concentration had no significant effect on NE-specific intestinal lesion scores in either experiment. Irrespective of the dietary treatment, a large number of birds had a thin and friable intestinal wall, with congested serosa with blood-engorged mesenteric vessels. Some birds had focal patches of hemorrhagic lesions in various intestinal regions, more frequently in the distal jejunum, proximally ileum, and cecal tonsils. However, typical field-type lesions specific to NE were not observed in any of the birds in either experiment. Although Met source or concentration had no significant effect on intestinal lesion scores, there was a trend of decreasing lesions with an increase in Met concentration, which corresponds well with C. perfringens colonization in these birds.


Figure 1
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  		Figure 1. Mean necrotic enteritis (NE) lesion scores in experiments 1 and 2 in 28-d-old broiler chickens given experimental diets (d 14 to 28) containing different levels or sources of Met. Bars represent mean ± SEM, n = 12. Lesion were scored on a 0 to 4 scale, where 0 = no intestinal lesion (apparently normal); 0.5 = severely congested serosa and mesentery engorged with blood; 1 = thin-walled and friable intestines with small red petechiae (> 5); 2 = focal necrotic lesions; 3 = patches of necrosis (1 to 2 cm long); and 4 = diffused necrosis typical of field cases.

 
Histological examination of formalin-fixed intestinal tissues from 28-d-old broiler chickens euthanized in both experiments revealed no lesions that were highly suggestive of NE. The lesions included only slight edema and diffuse hemorrhages in the lamina propria in some sections. No visible signs of necrosis or desquamation of epithelial cells in the villi were reported in either experiment. There was no evidence of gram-positive, rod-shaped organisms attached to the intestinal mucosa. Polymorphonuclear cells (PMN) infiltration in the lamina propria was not seen in any of the sections. We found no evidence of coccidial oocysts in any of the sections examined.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Evidence suggests that the amino acid composition of various protein sources is an important determinant of intestinal microbial growth in broiler chickens (Dahiya et al., 2006). Excessive concentrations of some amino acids might have a toxic effect on some bacterial species. Wilkie (2006) demonstrated a reduced growth of C. perfringens in the presence of Met in vitro. In one of his studies, he demonstrated that C. perfringens counts were virtually reduced to zero in minimal salt media supplemented with 10 mg/mL of DL-Met after a 24-h incubation. Hence, the current study was designed to determine whether 2 commonly used Met sources would have similar antibacterial effects against C. perfringens in vivo.

The overall performance of the birds was relatively poor in both experiments, which might be because the birds were under stress from the C. perfringens challenge and had high C. perfringens populations in their intestinal tract, which is evident from Tables 4Go and 5Go. The average feed consumption and BW gain were lower and the feed:gain ratio was comparatively poor in experiment 2 during the 21- to 28-d period, which might be related to the higher C. perfringens counts in experiment 2 compared with experiment 1. As evidenced by the intestinal lesion scores and high intestinal colonization of C. perfringens, most of the birds in the present study had subclinical NE, as documented by Lovland and Kaldhusdal (2001). Decreased growth rate and poor feed conversion efficiency have been reported previously in broilers having high numbers of C. perfringens in the intestinal tract (Stutz and Lawton, 1984; Kaldhusdal and Hofshagen, 1992; Dahiya et al., 2005, 2007).

Numerous studies have been conducted to compare the efficacy of DL-Met and liquid MHA-FA in broiler chickens (see, for example, Rostagno and Barbosa, 1995; Lemme et al., 2002). Some of these studies have been inconsistent in the value assigned to the efficacy of MHA-FA for a number of reasons (Maenz and Engele-Schaan, 1996; Drew et al., 2003). Drew et al. (2003) compared the apparent absorption of 3H-labeled L-Met with MHA-FA in germ-free and conventionally reared broiler chickens and observed a significantly lower residual MHA-FA in the distal ileum of germ-free birds than in conventional birds, in which there was no difference in residual Met level. Questions remain regarding the effect of these 2 Met sources on the intestinal microbial ecology of the birds. To our knowledge, this is the first study in which the direct impact of these 2 Met sources on the intestinal microflora of broiler chickens has been examined.

Clostridium perfringens populations were higher in the ileum and cecum of birds in experiment 2 than in experiment 1. This result might be because in experiment 2, C. perfringens was cultured in cooked meat medium, which supports C. perfringens growth and {alpha} toxin production much better than brain heart infusion medium, which was used in experiment 1 (J. P. Dahiya, personal communication). In the present study, we observed a significant reduction in C. perfringens growth with Met supplementation in the cecum (experiment 1), or in both the ileum and cecum (experiment 2). However, there were no significant differences between the 2 Met sources. We could find no literature on the toxic effects of high concentrations of Met on C. perfringens in vivo. Earlier, Muhammed et al. (1975) reported that Ala, Asp, and Met were stimulatory for the growth and sporulation of C. perfringens in vitro, but we cannot extrapolate the results from in vitro to in vivo because the conditions are entirely different in the gastrointestinal tract. There are differences in amino acid and vitamin requirements among various strains of C. perfringens (Fuchs and Bonde, 1957). Fuchs and Bonde also reported a significant increase in C. perfringens growth in the presence of Gly, Lys, and Ser and proved that not only individual amino acids but also the balance of amino acids is important for maximum growth of C. perfringens. There might be antagonism between amino acids (i.e., the action of certain amino acids being prevented by several other amino acids). Previously, we found that the amino acid Gly supports C. perfringens growth and {alpha}-toxin production both in vitro and in vivo (Dahiya et al., 2005, 2007; Wilkie et al., 2005).

The Met levels used in this study ranged to 2 to 4 times higher than those commonly used in commercial poultry diets. The supranutritional concentrations of Met were associated with reduced C. perfringens growth in broiler chickens; however, it might not be a commercially viable method of controlling NE because of the high cost of crystalline Met. The mode of action by which Met influences the intestinal populations of these important groups of bacteria in broiler chickens is unclear. Not all bacteria have the ability to utilize DL-Met or MHA-FA. Because C. perfringens is strictly dependent on carbohydrates, the effect of higher levels of dietary Met on intestinal C. perfringens populations might be indirect.

In experiment 2, we observed a significant elevation in lactobacillus population in the cecum of birds fed 0.8% Met-supplemented diets compared with the rest of the diets. In contrast to this, Streptococcus group D populations were decreased with Met supplementation in the ileum (experiment 2) and cecum (experiment 1). Hofshagen and Kaldhusdal (1992) reported a higher population of lactobacilli in the intestinal tract of broiler chickens fed corn-based diets and hypothesized that higher lactobacillus colonization impeded the development of NE. This effect might be indirect through the inhibition of C. perfringens colonization. We observed substantially lower populations of coliforms than lactobacilli and streptococci in the intestines of chickens, which is consistent with the findings of Barnes et al. (1972) and Stutz et al. (1983) and opposed to the findings of Hofshagen and Kaldhusdal (1992), who reported similar levels of these bacterial species. This discrepancy may be attributed to variations in bacteriological isolation procedures, feed composition, feed antibiotics, or environmental conditions among the studies.

Hegedus et al. (1993) documented that some lactic acid-producing bacteria (Lactobacillus plantarum, Leuconostoc mesenteriodes, Lactobacillus casei) have the ability to utilize DL-Met for their growth. Lactic acid bacteria have been characterized for their antagonistic action against Salmonella and Campylobacter in poultry (Gusils et al., 2003; Lan et al., 2003). Fukata et al. (1991) reported that the pathogenic effect of C. perfringens could be reduced by feeding chicks a monoflora of Lactobacillus acidophilus or Streptococcus faecalis. It is also possible that the proteins or amino acids are serving as an energy or nitrogen source for other bacterial species, which in turn modify the intestinal environment in a favorable way for lactobacillus proliferation.

In both experiments, despite inoculation of very high doses of C. perfringens, the mortality was low (2.38 and 3.57%, respectively), and necropsy examinations revealed that the deaths were not NE specific. This is in agreement with some previous studies in which chickens challenged with C. perfringens failed to induce mortality or other signs of NE, even though high colonization of C. perfringens was reported in the intestinal tract of the birds (Craven, 2000; Pedersen et al., 2003). In contrast to this, Al-Sheikhly and Truscott (1977) and Vissiennon et al. (2000) were able to induce various pathological changes and mortality in chickens when inoculated orally with C. perfringens directly into the duodenum. It is therefore possible that some of the vegetative cells are inactivated by the acidic pH in the gizzard when given orally.

In the present study, we observed a decreasing trend of NE intestinal lesions with a corresponding increase in dietary Met concentration. The demonstration of a relationship between NE lesion scores, performance data, and C. perfringens numbers is an important feature of both experiments. Al-Sheikhly and Truscott (1977) and Vissiennon et al. (2000) suggested that high C. perfringens populations and slight intestinal damage were apparently necessary for disease production when a broth culture was used. Earlier, Tanya et al. (2005) demonstrated a reduction in intestinal NE lesion scores in broiler chickens fed low-protein diets (CP 18%).

In contrast to some earlier findings, the microscopic lesions in the intestine were not conclusive of NE in the current study (Shane et al., 1985; Kaldhusdal et al., 1995). The presence of slight edema and hemorrhages in the lamina propria was observed in many birds. However, desquamated epithelial cells and PMN cells were not detected in any of the sections. Bryant et al. (1993) and Stevens et al. (1997) also demonstrated an absence of PMN cells at the site of C. perfringens infection. In the absence of a host response (suppression of PMN influx), clostridia proliferates rapidly, leading to local accumulation of toxins. Higher in situ concentrations of C. perfringens toxins, especially {alpha} toxin, further inhibit PMN influx and reach concentrations sufficient to cause membrane destruction (Stevens and Rood, 2000). Hence, in spite of high numbers of C. perfringens in the intestinal tract of these birds, the clinical disease could not be produced.

The results of the present study demonstrated that both Met sources might have some antibacterial effect against C. perfringens. Thus, it might be possible to inhibit C. perfringens growth in the intestinal tract of broiler chickens and prevent the occurrence of NE outbreaks through supplementation of low-protein diets with relatively high amounts of Met. Feeding low-CP diets supplemented with crystalline amino acids might be beneficial in terms of the growth of various enteric pathogens. Because there are some limitations to the culture-based methods for bacterial enumeration used in the present study, it would be interesting to use culture-independent approaches, such as denaturing gradient gel electrophoresis, in studying a wide group of microbiota with respect to different Met sources. Further studies to determine which bacterial species are involved in competition with the host for these 2 Met sources would be interesting.


   ACKNOWLEDGMENTS
 
The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (Ottawa, Ontario, Canada) and Degussa Corporation. The technical assistance of Darryl Wilkie and Jason Marshall is greatly appreciated.

Received for publication March 28, 2007. Accepted for publication July 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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