Abstract
Necrotic enteritis is a worldwide poultry disease caused by the overgrowth of Clostridium perfringens in the small intestine. An experiment with a 2×2 factorial design (supplementation with or without 40 mg lysozyme/kg diet for chickens challenged with or without C. perfringens) was conducted to investigate the inhibitory efficacy of exogenous lysozyme against intestinal colonization by C. perfringens in chickens subject to oral inoculation of C. perfringens type A on days 17 to 20. The C. perfringens challenge resulted in significant increase of C. perfringens, Escherichia coli and Lactobacillus populations in the ileum, bacteria translocation to the spleen, the intestinal lesion scores , There was significantly lower intestinal lysozyme activity in the duodenum and jejunum and weight gain during days 14 to 28 of the experiment. The addition of exogenous lysozyme significantly reduced the concentration of C. perfringens in the ileum and the intestinal lesion scores, inhibited the overgrowth of E. coli and Lactobacillus in the ileum and intestinal bacteria translocation to the spleen, and improved intestinal lysozyme activity in the duodenum and the feed conversion ratio of chickens. These findings suggest that exogenous lysozyme could decrease C. perfringens colonization and improve intestinal barrier function and growth performance of chickens.
Introduction
Necrotic enteritis (NE) is a worldwide poultry disease (Engström et al., Citation2003) and has significant economic impact on the poultry industry (Van der Sluis, Citation2000). The disease causes severe necrosis of the intestinal tract and high mortality in acute clinical cases, and affects growth rate and feed efficiency in sub-clinical cases in birds (Hofshagen & Kaldhusdal, Citation1992; Wages & Opengart, Citation2003). It is well known that proliferation of the enteric bacterium C. perfringens type A or C and production of extracellular toxins are the major factors for NE. However, many cofactors are usually required to promote overgrowth of C. perfringens in the intestinal tract and precipitate an outbreak of NE, including management and environmental conditions (Craven et al., Citation2001), co-infection with coccidia (Collier et al., Citation2008), diet composition such as wheat and barley (Riddell & Kong, Citation1992; Langhout, Citation1998) and diet form (pelleted versus mash) (Engberg et al., Citation2002).
At present, the most effective strategy to control C. perfringens-associated NE in poultry is using in-feed antibiotics, such as virginiamycin, bacitracin and tylosin. However, due to the possible negative impact of antibiotics on the environment and human health, banning of antibiotics has stimulated interest in finding alternative compounds from natural sources to control the incidence and severity of NE in the post-antibiotic era.
Lysozyme (EC 3.2.1.17, muramidase), a natural antimicrobial protein, occurs in a number of animal secretions and is considered an important component of the innate immune system. It exerts bacteriolytic activity by hydrolysing the β-1,4-glycosidic linkage between N-acetlymuraminic acid and N-acetylglucosamine of bacterial cell wall peptidoglycan and is most effective against many Gram-positive bacteria (Phillips, 1966). Because of its abundance in egg white, lysozyme is commercially extracted from hen eggs and has been applied as a natural food preservative (Losso et al., Citation2000) and a therapeutic drug for humans (Sava, Citation1996).
Lysozyme is efficacious for the treatment of infectious diseases in humans and animals after administration by oral, intravenous, intraperitoneal and topical routes. For example, oral administration of egg white lysozyme is now used clinically in human medicine for the therapy of inflammatory diseases of respiratory and digestive epithelia (Seno et al., Citation1998). In rainbow trout, lysozyme injections decreased mortality from challenge with Aeromonas salmonicida by more than threefold and oral administration decreased mortality from infectious pancreatic necrosis virus twofold (Siwicki et al., Citation1998). Intraperitoneal administration decreases the pathology resulting from a Klebsiella pneumoniae infection in mice (Ivanovska et al., Citation1996). However, there are few reports on the effect of lysozyme on poultry disease. Recently, Zhang et al. (Citation2006) reported the antimicrobial activity of lysozyme against isolates of C. perfringens type A associated with broiler NE in vitro, and thought that lysozyme could kill the bacterial pathogen. However, no cage and floor pen trials using an appropriate challenge model have been conducted to further evaluate the efficacy of lysozyme in vivo against C. perfringens colonization.
The present study used a chicken model of C. perfringens challenge to determine whether the inhibitory efficacy of exogenous lysozyme against intestinal colonization by C. perfringens exists. The effects of lysozyme on growth performance, intestinal lesion scores, intestinal microbial populations relative to colonization by C. perfringens, intestinal barrier function, and intestinal lysozyme activity and expression in chickens were investigated.
Materials and Methods
Experimental treatment
One hundred and sixty-eight 1-day-old male Arbor Acre broiler chickens were randomly allocated into eight replicates (cages) of 21 birds each. From day 1 until day 14 of the experiment, birds were provided with the same diet. On day 14, birds were weighed and randomly reassigned to four treatments in a design with a 2×2 factorial arrangement; that is, supplementation with or without 40 mg lysozyme/kg diet for birds challenged with or without C. perfringens. Each treatment had six replicates (cages) with seven birds each. The feeding period lasted 28 days. The chickens had free access to feed and water and were housed in wire cages and maintained on a 24-h constant-lighting programme. An unmedicated maize–wheat–soybean meal diet was formulated to meet or exceed National Research Council (Citation1994) requirements. Composition of the diet and nutrient levels are presented in .
Table 1. Composition of the diet and nutrient levels
C. perfringens challenge
C. perfringens challenge was based on the model originally developed by Dahiya et al. (Citation2005), with some modifications. Briefly, a chicken C. perfringens type A field strain isolated from a clinical case of NE was obtained from China Veterinary Culture Collection Center. The organism was cultured anaerobically on blood agar base containing 5.0% sheep blood and 100 mg/l neomycin sulphate for 18 h at 3°C, and then aseptically inoculated into cooked meat medium and incubated anaerobically overnight at 37°C. On days 17 to 20, all birds (except control and lysozyme treatment birds) were orally gavaged once per day with this actively growing culture of C. perfringens type A (2.0×108 colony-forming units/ml, 1.0 ml/bird). Based on previous studies, sampling was subsequently performed on day 22 when the signs of NE are most severe and on day 28 when recovery has occurred (Hofacre et al., Citation1998; Collier, et al., Citation2008).
Pathological variables
Birds were observed on a cage basis at least once daily for any signs of NE (e.g. huddling, diarrhoea, depression, or mortality), and all dead birds were necropsied to determine the cause of death. Six birds per treatment were killed by cervical dislocation and necropsied on days 22 and 28. The duodenum and jejunum (from the gizzard to the 10 cm preceding Meckel's diverticulum) were examined and intestinal lesions were scored on a scale of 0 to 4 as described previously by Dahiya et al. (Citation2005); that is, 0 = normal intestinal appearance; 0.5 = severely congested serosa and mesentery engorged with blood; 1 = thin walled and friable intestines with small red petechiae; 2 = focal necrosis, grey appearance and small amounts of gas production; 3 = sizable patches of necrosis, gas-filled intestine and small flecks of blood; and 4 = severe extensive necrosis, marked haemorrhage, large amounts of gas in the intestine.
Growth performance
Feed consumption and body weight for each replicate (cage) were recorded for the period of days 14 to 28 to calculate the feed conversion ratio (FCR). When calculating the FCR, the body weight of dead birds was taken into account.
Intestinal barrier function
Twelve birds per treatment were sacrificed on days 22 and 28 to evaluate barrier function of the intestinal mucosa. The barrier function was determined by the indirect method of bacterial translocation (BT) to the liver and spleen. Total aerobic and anaerobic bacteria were quantified from the liver and spleen by standard aerobic and anaerobic cultivation techniques on mixed media agar plates. The specimen was considered positive when more than 100 colony-forming units/g organs were present.
Enumeration of ileum bacteria
On days 22 and 28, the fresh ileum (Meckel's diverticulum to 1 cm proximal to the ileo-caecal junction) contents from six birds per treatment were collected aseptically into sterilized plastic dram vials and mixed well. Using a sterile spatula, the samples were transferred into preweighed 15-ml sterile plastic tubes, weighed and diluted in peptone water to an initial 10−1 dilution for the enumeration of C. perfringens, Escherichia coli, Lactobacillus and Bifidobacterium. The samples were kept on ice until plated within 3 h of collection. The 10-fold dilutions were spread in duplicate on a blood agar base (Land Bridge Technology Ltd, Beijing, China) containing 5.0% sheep blood and 100 mg/l neomycin sulphate for the enumeration of C. perfringens, MacConkey agar (Land Bridge Technology Ltd) for the enumeration of E. coli, MRS agar (Land Bridge Technology Ltd) for the enumeration of Lactobacillus, and Wilkens-Chalgren agar (Oxoid Ltd, Basingstoke, UK) for the enumeration of Bifidobacterium. C. perfringens was incubated anaerobically for 24 h at 37°C, Lactobacillus and Bifidobacterium were incubated anaerobically for 48 h at 37°C, and E. coli was incubated aerobically for 24 h at 37°C.
Intestinal lysozyme activity assay
Samples for assay of intestinal lysozyme level were prepared by obtaining intestinal flushes. Six chickens from each treatment were fasted overnight to reduce lumen contents solely for intestinal lysozyme activity assay, and then 10 g duodenum, jejunum and ileum were collected from each bird and flushed with an equal amount of saline (2 ml), respectively. Intestinal flushes were collected and centrifuged for 5 min at 1253×g, and supernatants were decanted and maintained at −70°C. The lysozyme level of supernatants was determined according to the method of Kreukniet et al. (Citation1994) with some modifications. A series of concentrations (1.5, 2.0, 3.0, 4.0 and 6.0 µg/ml) of crystalline lysozyme (Sigma Chemical Co., Saint Louis, Missouri, USA) dissolved in phosphate buffer (pH 6.2) was used to make the standard curve. A solution of Micrococcus lysodeikticus (Institute of Microbiology, Chinese Academy of Science, Beijing) was added to the standard dilution to determine lysozyme level.
Lysozyme mRNA expression assay
Total RNA was extracted from duodenum tissue from six 22-day-old birds per treatment using Trizol reagent (Invitrogen Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions. The total RNA sample was reverse transcribed using the RevertAid First Strand cDNA Synthesis kit (MBI Fermentas, Hanover, Maryland, USA) according to the manufacturer's instructions. Reverse transcription was performed at 42°C for 1 h followed by heat inactivation for 5 min at 70°C. All of the cDNA preparations were stored frozen at −20° until further use.
A quantitative polymerase chain reaction (PCR) assay was performed with the 7900-HT fluorescence detection system (Applied Biosystems, Foster City, California, USA) according to optimized PCR protocols using the SYBR-Green qPCR kit (Applied Biosystems). To amplify lysozyme g and g2 and β-actin cDNA fragments, the following sequences of PCR primers pairs were used: forward, 5′-CACGCTGGCAAAATACTGAAG-3′ and reverse, 5′-TTCCCAACACCAGCATTGTAG-3′ for lysozyme g (X61002); forward, 5′-CATTCCATCTTTGGTTGC-3′ and reverse, 5′-CCACCTTTGAGCTGCTGTTC-3′ for lysozyme g2 (Nile et al., Citation2004); and forward, 5′-CCACCGCAAATGCTTCTAAAC-3′ and reverse, 5′-AAGACTGCTGCTGACACCTTC-3′ for β-actin (NM_205518). cDNA was amplified with an initial 1-min denaturation step of 95°C, 40 cycles of 1 min at 95°C, 1 min at the annealing temperature 55°C, 2 min of 72°C and a final extension step of 72°C for 10 min.
Relative standard curve methods were used for quantification of gene expression. Copy numbers were determined from two independent cDNA preparations of each sample and were calculated relative to a dilution series of the respective reference plasmids, comprising 103 to 108 copies. The reference plasmids contained cloned reverse transcriptase-PCR products obtained with these primers. The housekeeping gene, β-actin, was used as the internal standard for the PCR reaction. The number of cycles halfway through the experimental phase was determined and was used to calculate the relative expression level compared with a known β-actin standard.
Statistical analysis
The BT incidence was evaluated by Fisher's exact test of SPSS (version 12.0). Statistical analysis of other data was performed by two-factorial analysis of variance with SPSS. Individual treatment means were compared using Duncan's multiple comparison when the significant (P<0.05) interaction between the main effects was observed; differences between the means were considered significant when P<0.05.
Results
Clinical signs and necrotic enteritis lesion
With the challenge of C. perfringens, most of the birds initially became dull and depressed, and had abnormally wet droppings where undigested feed was detectable. Some birds had very thin intestinal walls with congested mucosa and mesenteric vessels engorged with blood, and had focal haemorrhagic lesions in the duodenum and jejunum. However, typical field type lesions specific to NE were not observed in any of the birds, and no death occurred during the experiment. Intestinal lesion scores are presented in . No intestinal lesion was observed in the birds without C. perfringens challenge, and C. perfringens challenge significantly increased (P <0.05) intestinal lesion scores on days 22 and 28. The intestinal lesion scores of birds challenged with C. perfringens were higher on day 22 than on day 28. The main effect of lysozyme and its interactive effect with C. perfringens challenge on lesion scores were not significant (P =0.078) on day 22. On day 28, the interactive effects were found between lysozyme and C. perfringens challenge for the lesion scores, and the addition of lysozyme significantly reduced the severity of intestinal lesion of birds challenged by C. perfringens (P <0.05).
Table 2. Growth performance and intestinal lesion scores of broiler chickens
Growth performance
On day 14, the initial body weight of each treatment was analysed and the difference was not significant (P >0.05) (). Analysis of the main effects in showed that C. perfringens challenge resulted in a significant decrease in weight gain (P <0.05) and a tendency to increase the FCR (P =0.064), whereas it did not affect feed intake of birds during days 14 to 28 of age. Supplemental lysozyme significantly improved the FCR (P <0.05) and tended to increase weight gain (P =0.082).
Microbial populations of ileum digesta
The microbial diversity of the ileum dramatically changed when birds were challenged with C. perfringens (). Twenty-two-day-old chickens challenged with C. perfringens had the greater number of C. perfringens, E. coli, Lactobacillus and Bifidobacterium in the ileum compared with 28-day-old chickens. On days 22 and 28, a sharp increase of C. perfringens, E. coli, and Lactobacillus counts was observed following challenge with C. perfringens (P <0.05), whereas their populations were decreased significantly by dietary lysozyme (P <0.05). There was a significant interaction between dietary lysozyme and C. perfringens challenge on C. perfringens counts (P <0.05) on day 28. The number of C. perfringens was the lowest in birds without challenge and was the highest in the birds with C. perfringens challenge but without lysozyme. No influence was observed on the populations of Bifidobacterium by the main effects and their interaction on days 22 and 28 (P >0.05).
Table 3. C. perfringens, E. coli, Lactobacillus and Bifidobacterium. populations (log10 colony-forming units/g) in the ileum of broiler chickens
Bacterial translocation
The data of the intestinal mucosa barrier function are presented in . Aerobic and anaerobic bacteria were observed in the spleen and liver for all birds with C. perfringens challenge. BT was also detected in the spleen of birds without C. perfringens challenge, which reflected the immaturity of barrier function at this early age. Compared with the control and lysozyme-supplemented birds, the C. perfringens-challenged birds had greater (P <0.05) liver aerobic BT rates on day 22 and greater spleen anaerobic and aerobic BT rates on both days 22 and 28. The C. perfringens-challenged birds with lysozyme had lower (P <0.05) spleen aerobic BT rates on day 22 and lower spleen anaerobic and aerobic BT rates on day 28 compared with the C. perfringens-challenged birds without lysozyme. No significant difference (P >0.05) was noted, however, between the two groups with respect to liver anaerobic and aerobic BT rates on days 22 and 28.
Table 4. Bacteria translocation rates of the liver and spleen (%) of broiler chickens
Lysozyme level and mRNA expression
As shown in , C. perfringens challenge significantly reduced (P <0.05) lysozyme activity in the duodenum and jejunum on days 22 and 28. Lysozyme treatment had a tendency (P =0.055) to increase lysozyme activity in the duodenum on day 22. On day 28 there was a significant interaction (P <0.05) between lysozyme treatment and C. perfringens challenge on lysozyme activity in the duodenum. The birds with only C. perfringens challenge had the lowest lysozyme activity in the duodenum, and the exogenous lysozyme supplementation significantly increased (P <0.05) lysozyme activity in the duodenum of birds challenged with C. perfringens. No significant influence (P >0.05) was observed on lysozyme activity in the ileum by the main effects and their interaction on days 22 and 28.
Table 5. Lysozyme activity (mg/l) in the duodenum, jejunum and ileum of broiler chickens
The expression of lysozyme g and g2 mRNA in the duodenum was not affected by C. perfringens challenge, lysozyme supplementation and their interaction ().
Table 6. Lysozyme g and g2 mRNA relative content in the duodenum of broiler chickens on day 22
Discussion
In the present trial, C. perfringens challenge did not result in overt clinical signs of NE and death of birds, but many of the challenged birds did show distinctly pronounced pathological changes (hyperaemia and haemorrhages) and high colonization of C. perfringens in the intestinal tract, as documented by some researchers (Kaldhusdal & Hofshagen, Citation1992; Craven, Citation2000; Pedersen et al., Citation2003; Olkowski et al., Citation2006; Dahiya et al., Citation2007). Their controlled studies of chickens challenged with high doses of C. perfringens have also failed to induce mortality or other signs of NE. Factors such as coccidiosis, infectious bursal disease, or dietary stress would favour an outbreak of NE experimentally in chickens with inoculation of high doses of C. perfringens (Ficken & Wages, Citation1997).
Although the challenge of high doses of C. perfringens does not always induce NE-specific mortality and clinical signs, it causes damage to the intestinal mucosa to lead to decreased growth rate and poor feed conversion efficiency of chickens (Kaldhusdal & Hofshagen, Citation1992; Dahiya et al., Citation2005, 2007), which reportedly results in the greatest economic losses in the poultry production industry (Dahiya et al., Citation2006). The intestinal lesions associated with C. perfringens challenge are the result of C. perfringens proliferation and protein toxins released by the bacteria in intestinal tract. The most recently discovered toxin, NetB toxin, is now known to have a major role in the intestinal lesions in poultry (Keyburn et al., Citation2008), whereas other toxins—such as α-toxin, which is believed to be the major virulence factor in early studies (Fukata et al., Citation1988; Lovland et al., Citation2004; Kulkarnia et al., Citation2007)—play no direct part in pathogenesis of NE (Keyburn et al., Citation2006; Van Immerseel et al., Citation2008). Destruction of mucosal tissue by protein toxins manifests as macroscopic lesions in the jejunum and ileum but also in the duodenum (Truscott & Al-Sheikhly, Citation1977; Fukata et al., Citation1991). Intestinal lesions disturbing the interface between enterocytes and lamina propria induce strong inflammatory response in birds (Titball et al., Citation1999; Olkowski. et al., Citation2006; Collier et al., Citation2008), assuredly impact normal digestive function, impair nutrient utilization and, therefore, suppress performance of birds. In this study, growth performance of birds was suppressed greatly, and macroscopic lesions were observed mainly in the duodenum and jejunum after C. perfringens challenge. On day 28, significantly reduced FCRs and intestinal lesion scores of birds supplemented with lysozyme indicate that lysozyme greatly improved the performance and gut health of birds challenged with C. perfringens with the time prolongation of its administration.
The exact mechanism(s) by which lysozyme alleviates the intestinal lesions of chickens is unclear, but it is suggested to correlate with the directly inhibitive effects of lysozyme on C. perfringens proliferation and its immunomodulating activity. Lysozyme's ability to inhibit several other Clostridium species, including Clostridium tyrobutyricum, Clostridium thermosaccharolyticum and Clostridium botulinum, is documented by Carini et al. (Citation1985) and Hughey & Johnson (Citation1987). Zhang et al. (Citation2006) reported that lysozyme in vitro could not only kill C. perfringens, but could also reduce production of α-toxin, the causative agent for intestinal lesions. In our study, the production of α-toxin was not determined; however, this does not rule out the possibility of a suppression of α-toxin production due to the significant reduction of the C. perfringens number in the ileum.
Many previous investigators reported the immunomodulating activity of lysozyme in humans and animals (Sava, Citation1996). In vitro, lysozyme stimulates immunoglobulin production of specific human–human hybridoma line and human peripheral blood lymphocyte (Murakami et al., Citation1997; Sugahara et al., Citation2000). Moreover, lysozyme is administrated orally and absorbed in the gut to increase plasma levels of lysozyme, which could produce systemic effects and potentiate the activity of monocytes and macrophages (Seno et al., Citation1998). At the intestinal level, lysozyme has been reported to interact with intestinal bacteria to liberate immune-modulating peptidoglycans and Peyer's patches, and intraepithelial lymphocytes to activate the host's immune system (Jollès, Citation1976; Namba et al., Citation1981). Therefore, it is speculated that lysozyme could activate systemic and intestinal immune responses to reduce the intestinal lesions of birds challenged with C. perfringens.
Initial physiological changes caused by NE include less intestinal motility, lower intestinal pH, and an increase in intestinal protein concentration due to malabsorption or leakage of serum proteins into the lumen of the intestine or both (Shane et al., Citation1985), which are associated with the occurrence of bacterial overgrowth (Eamonn et al., Citation2006). Its presence could result in mucosal injury and malnutrition. Many populations of bacteria have been shown to coexist with NE, and one of the largest of them is E. coli (Collier et al., Citation2003, 2008; McReynolds et al., Citation2004). In the present study, with sharp increases in the C. perfringens population there was a corresponding increase in E. coli populations in the ileum, which is consistent with the report of McReynolds et al. (2004). In addition, C. perfringens challenge also induced the significant increase of Lactobacillus populations in the ileum, which was postulated as an antagonistic effect against C. perfringens and E. coli (Mangell et al., Citation2002). With the antimicrobial activity of lysozyme, its administration reduced the C. perfringens, E. coli and Lactobacillus populations in the ileal digesta of birds with and/or without C. perfringens challenge.
Lysozyme has bacteriostatic and bactericidal activity against many Gram-positive bacteria and had only a limited ability to affect Gram-negative bacteria. Enzymatic hydrolysis of lysozyme has been found to enhance its activity, by exposing antibacterial portions of the protein (Pellegrini et al., Citation1997; Ibrahim et al., Citation2001), and producing antibacterial peptides (Mine et al., Citation2004; Pellegrini et al., Citation2000). Peptides corresponding to amino acid residues 98 to 108, residues 15 to 21 (Mine et al., Citation2004) and residues 98 to 112 (Pellegrini et al., Citation2000) possessed antimicrobial activity against E. coli and Staphylococcus aureus. In addition, lysozyme usually functions in association with a complement, lactoferrin, sIgA and other microbicidal compounds (Branen & Davidson, Citation2004). The present results, which showed that lysozyme reduced E. coli and Lactobacillus counts in the ileal digesta of birds, suggest that digestive enzymes and other compounds such as sIgA in the gut may have induced the novel antimicrobial activity and broadened the spectrum of activity of lysozyme in vivo. In addition, the apparent effect of lysozyme on E. coli and Lactobacillus counts could also be indirect in those birds challenged with C. perfringens; that is, via its inhibitory effect on C. perfringens colonization reducing their counts.
BT is defined as the passage of viable bacteria from the intestinal tract through the epithelial mucosa into extra-intestinal organs. Various studies have shown that bacterial translocation is a function of the integrity of the intestinal epithelium, bacterial overgrowth, and the status of the host immune system (Baker et al., Citation1987; Berg, Citation1995). Impaired mucosal surfaces and immune defence mechanisms can increase vulnerability of the intestinal epithelium with an augmented risk of bacterial and viral penetration, or bacterial overgrowth in the intestine. In the current study, C. perfringens challenge led to impaired intestinal mucosa and high numbers of C. perfringens and E. coli, which could account in part for the high anaerobic and aerobic BT rates in the spleen and liver of birds challenged with C. perfringens. Our findings suggest that lysozyme administration reduced the incidence of spleen anaerobic and aerobic BT of birds challenged with C. perfringens to a great extent because of its positive effects on the improvement of intestinal lesions and the inhibition of the bacterial overgrowth.
The secretion of lysozyme in the small intestine is believed to control the overgrowth of normal flora and pathogens. Two structurally distinct forms of lysozyme are found in vertebrates, chicken-type (c) and goose-type (g) (Short et al., Citation1996). In fish, lysozyme g is present in macrophages, neutrophils, and eosinophilic granular cells in skin epidermis and intestine (Fletcher & White, Citation1976; Sveinbjørnsson et al., Citation1996). In higher vertebrates, lysozyme c is present in polymorphonuclear and mononuclear phagocytes, and in Paneth cells, which are specialized intestinal epithelial cells (Keshav et al., Citation1991; Dohrman et al., Citation1994). Theisen et al. (Citation1986) and Short et al. (Citation1996) reported that the chicken genome, like the human, has a single lysozyme c gene, which is highly expressed in the oviduct and macrophages. Nile et al. (Citation2004) found that three lysozyme genes were expressed in the chicken small intestine by villous enterocytes to fulfil complimentary roles in protecting the intestine, lysozyme c in 4-day-old but not older chicken intestine, lysozyme g in 4-day-old to 35-day-old chicken intestine, and a novel chicken lysozyme g2 in the intestine from chickens of all ages. To our knowledge, there are no reports about the effects of C. perfringens challenge and lysozyme administration on the lysozyme activity and gene expression in the intestine. In the present study, C. perfringens challenge reduced lysozyme activity in the duodenum, suggesting that activated lysozyme deficiency may facilitate the bacterial infections. This modification could be the result of the inactivation of partial lysozyme or the decrease of lysozyme secretion due to the sharp increase of C. perfringens in the gut. Exogenous lysozyme administration increased the intestinal lysozyme activity with C. perfringens challenge, but it did not influence that in non-challenged birds, which could enable speculation that the effects of exogenous lysozyme on intestinal lysozyme activity are associated with C. perfringens colonization in the gut. Lysozyme g and g2 gene expression was analysed in 22-day-old chicken duodenum because the significant difference of the lysozyme level in the chicken duodenum existed on day 22 after C. perfringens challenge and/or lysozyme treatment. Intestinal lysozyme g and g2 mRNA expression was not changed by exogenous lysozyme treatment and C. perfringens challenge. However, further analyses must be undertaken to determine whether the translation of the two proteins was changed by exogenous lysozyme treatment and C. perfringens challenge.
In conclusion, exogenous lysozyme administration could decrease C. perfringens colonization, intestinal mucosal lesion and intestinal bacteria translocation, reduce high levels of ileal E. coli and Lactobacillus, increase intestinal lysozyme activity, and improve production performance in broilers challenged with C. perfringens.
Acknowledgements
Supported by the project 2006BAD12B0F# of Eleventh Five Year Plan of Science and Technology of China.
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