In vivo and in vitro protective effect of arginine against intestinal inflammatory response induced by Clostridium perfringens in broiler chickens

Background: Necrotic enteritis is a widespread disease in poultry caused by Clostridium perfringens. We previously reported that dietary arginine supplementation protected the intestinal mucosa of broiler chickens with necrotic enteritis, but the related protective mechanisms remain unclear. The in vivo trial was designed as a 2 × 2 factorial arrangement to evaluated the effects of arginine supplementation on inflammatory responses, arginine transporters,arginine catabolism and JAK-STAT signalling pathway in broiler chickens challenged with C. perfringens or without C. perfringens. Furthermore, we validated the in vivo results using intestinal epithelial cells of chicken embryos.Results: C. perfringens infection markedly increased gut gross pathological and histopathological lesion scores,promoted liver C. perfringens invasion, reduced serum arginine levels, and elevated jejunal mucosal lysozyme activities(P 0.05), but these effects were significantly reversed by arginine supplementation in vivo(P 0.05). The challenge significantly increased serum procalcitonin levels, jejunal mucosal iNOS activities and jejunal IL-6, TGF-β3,cationic amino acid transporter(CAT)-1, and CAT-3 mRNA expression(P 0.05), whereas arginine supplementation significantly reduced jejunal IFN-γ, IL-1β, IL-6, IL-10, TGF-β3, and CAT-3 mRNA expression(P 0.05). Arginine supplementation significantly attenuated the C. perfringens challenge-induced increases in jejunal i NOS, arginase 2,arginine decarboxylase, arginine:glycine amidinotransferase, JAK1, JAK3, STAT1, and STAT6 mRNA expression(P 0.05).The in vitro experiment showed that C. perfringens challenge markedly increased cellular cytotoxicity and the mRNA expression of IL-1β, IL-8, IL-10, CAT-1 and CAT-3(P 0.05), which were significantly reversed by 50 μmol/L and/or400 μmol/L arginine pre-treatment(P 0.05).Conclusions: Arginine prevented C. perfringens challenge-induced circulated arginine deficiency, normalized intestinal arginine transport and catabolism, down-regulated JAK-STAT signalling pathway and attenuated the inflammatory response, which exerted protective effects on the intestine of broiler chickens.     

In vitro antibacterial activity of thymol and carvacrol and their effects on broiler chickens challenged with Clostridium perfringens

Background:In the post-antibiotic era,essential oils(EO) are promising alternatives to growth-promoting antibiotics.The aim of the present study was to investigate the antibacterial activities of an EO product and its components thymol and carvacrol in vitro,and the efficacy of EO to control Clostridium perfringens challenge in broiler chickens.Results:The in vitro minimum inhibitory concentration assay showed strong antibacterial activity of the EO product,thymol,and carvacrol against pathogenic Escherichia coli,C.perfringens,and Salmonella strains,and weak activity towards beneficial Lactobacillus strains.Besides,an additive effect was observed between thymol and carvacrol.The in vivo study was carried out with 448 male broiler chicks following a 4 × 2 factorial arrangement to test the effects of EO supplementation(0,60,120,or 240 mg/kg EO in wheat-based diet),pathogen challenge(with or without oral gavage of C perfringens from day 14 to day 20) and their interactions.Each treatment consisted of eight replicate pens(seven birds/pen).The challenge led to macroscopic gut lesions,and resulted in a significant increase in ileal populations of C.perfringens and Escherichia subgroup(P 0.05) on day 21.Dietary EO supplementation did not influence C perfringens numbers,but linearly alleviated intestinal lesions on day 21 and 28(P=0.010 and 0.036,respectively),and decreased Escherichia populations in ileum with increased EO dosages(P=0.027 and 0.071 for day 21 and 28,respectively).For caecum,EO quadratically influenced Lactobacillus populations on day 21(p=0.002),and linearly decreased the numbers of total bacteria and Escherichia on day 28(P=0.026 and 0.060,respectively).Mean thymol and carvacrol concentrations in the small intestine were 0.21 and 0.20 μg/g in intestinal digesta(wet weight),respectively,for birds fed 60 mg/kg EO,and 0.80 and 0.71 μg/g,respectively,for birds fed 240 mg/kg EO.Conclusions:These results indicated that dietary EO supplementation could affect intestinal microbiota and alleviate intestinal lesions in broilers,which may contribute in controlling C.perfringens infection in broiler chickens.     

In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens

Viable Clostridium perfringens ranging from 10(5) to 10(8) g-1 was detected in all of 88 intestinal content specimens of necrotic enteritis in broiler chickens. In vitro lecithinase activity and sensitivity to 22 antimicrobial agents were determined for the 88 isolates. The activities of lecithinase in the culture filtrate of isolates were estimated to be 0.5 to 4.0 AE ml-1 as alpha-antitoxin equivalent. With reference to antimicrobial activity penicillins and cephazolin showed excellent activity and no resistance; peptides, of the agents used as growth promoters, showed that all except bacitracin had low minimum inhibitory concentration levels (1.6 micrograms ml-1 or less) against this organism; polyethers of monensin, salinomycin and lasalocid were generally adequate in low concentrations while there was a high level of resistance to three tetracyclines in 90 per cent of the strains and all isolates were insusceptible to streptomycin of the aminoglycoside antibiotics.     

Characterisation and response of intestinal microflora and mucins to manno-oligosaccharide and antibiotic supplementation in broiler chickens

1. An experiment was conducted to characterise and evaluate, in comparison to zinc bacitracin (ZnB), the response of intestinal microflora and mucins to manno-oligosacchares (MOS, Bio-MOS®, Alltech Biotechnology, Nicholasville, Kentucky, USA).

2. Supplementation of MOS and ZnB selectively increased the intensity of sulphomucins. As revealed by the plate culture method, MOS accelerated the maturation of gut microflora by promoting the growth of lactobacilli in the ileal mucosa and vice versa on ileal and caecal clostridia. Unlike MOS, ZnB suppressed the growth of intestinal bacteria, especially those of lactobacilli and clostridia. Use of T-RFLP further revealed that MOS increased the diversity of lactobacilli in the ileum and ileal mucosa but the opposite was observed for ZnB. It also appears that MOS and ZnB possessed a common property in differentially favouring the growth of certain Lactobacillus species. There was also evidence to show that both MOS and ZnB also increased the homogeneity of the gut microflora, possibly through the regulation of the overall gut bacterial communities.

3. Improvement in intestinal microbial homogeneity and mucin synthesis, coupled with the differential selections for certain specific beneficial bacterial species, may ultimately be proven to be the target mechanisms in the search for more effective alternatives to antibiotics.     

Effect ofBacillus amyloliquefaciens-based direct-fed microbials and antibiotic on performance,nutrient digestibility,cecal microflora,and intestinal morphology in broiler chickens

Two hundred eighty-eight 1-d-old male Arbor Acres broilers were randomly allocated to 4 treatments to examine the effect of usingBacillus amyloliquefaciens-based direct-fed microbials (DFM) to replace antibiotics in the late growth stage on performance, nutrient digestibility, cecal microflora, and intestinal morphology in broilers. The experimental diets were (1) a negative control (NC) group fed a basal diet without any antibiotics; (2) a positive control (PC) group fed the basal diet with 200 mg/kg of zinc bacitracin; (3) a test group fed the basal diet with 200 mg/kg of zinc bacitracin from d 1 to 21 and 30 mg/kg of DFM from d 22 to 42 (DFM30); and (4) a test group fed the basal diet with 200 mg/kg of zinc bacitracin from d 1 to 21 and 60 mg/kg of DFM from d 22 to 42 (DFM60). The results showed that DFM30 and DFM60 increased BW gain and improved FCR as compared with NC group (P < 0.05). Generally, the PC, DFM30, and DFM60 groups showed increased apparent total tract nutrient digestibility for DM, CP, and AME as compared with the NC group. Both the DFM30 and DFM60 groups decreased cecalEscherichia coli population as compared with the NC group, and increased the population ofLactobacillus as compared with the PC group. The PC, DFM30, and DFM60 groups increased villus height and the ratio of villus height to crypt depth compared with NC. In conclusion, dietary supplementation withB. amyloliquefaciens-based DFM during d 22 to 42 may enhance performance by increasing nutrient digestibility and improving intestinal health in birds fed diets supplemented with zinc bacitracin from d 1 to 21.     

Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens

Background: Necrotic enteritis caused by Clostridium perfringens infection leads to serious economic losses in the global poultry production. In the present study, we investigated the protective effects of essential oils(EO, which contained 25 % thymol and 25 % carvacrol as active components) supplementation on growth performance, gut lesions, intestinal morphology, and immune responses of the broiler chickens infected with C. perfringens. A total of448 1-day-old male broiler chicks were allocated into eight treatment groups following a 4 × 2 factorial arrangement with four dietary EO dosages(0, 60, 120, or 240 mg/kg) and two infection status(with or without C. perfringens challenge from d 14 to 20).Results: The challenge did not impair the growth performance of birds, but induced gut lesions and increased crypt depth in the ileum(P ≤ 0.05). It also down-regulated the claudin-1 and occludin m RNA expression(P ≤ 0.05),up-regulated the m RNA expression of interleukin-1β(P ≤ 0.05), tended to increase the toll-like receptor(TLR)2 m RNA expression(P 0.10) in the ileum, and enhanced the mucosal secretory Ig A production(P ≤ 0.05). In the challenged birds, dietary EO supplementation linearly alleviated the gut lesions and improved the ratio of vil us height to crypt depth(P ≤ 0.05), and the supplementation of 120 and 240 mg/kg EO increased the serum antibody titers against Newcastle disease virus(P ≤ 0.05). Regardless of challenge, the EO supplementation showed a tendency to linearly elevate the feed conversion efficiency between 14 and 28 d of age as well as the occludin m RNA expression(P 0.10), and linearly inhibited the m RNA expression of TLR2 and tumor necrotic factor-α in the ileum(P ≤ 0.05).Conclusions: The dietary supplementation of EO could alleviate the intestinal injury by improving intestinal integrity and modulating immune responses in the C. perfringens-challenged broiler chickens.     

In vitro and in vivo protective effects of three mycotoxin adsorbents against ochratoxin A in broiler chickens

1. The objective of this study was to investigate in vitro and in vivo (in broiler chickens) ochratoxin A (OTA) adsorption efficiency of three different adsorbents: inorganic (modified zeolite); organic (esterified glucomannans) and mixed (inorganic and organic components plus enzymes).

2. The aim of the study was to investigate which of these adsorbents provided the best protection against the presence of residues of OTA in the pectoral muscle and liver of broilers given an OTA-contaminated diet. In addition, it was important to test and compare the results of adsorbent efficiency using two different in vitro methods.

3. The results from classical in vitro investigations carried out in the artificial intestinal fluid, showed that the inorganic adsorbent (Mz), exhibited the highest adsorption, having adsorbed 80.86 ± 1.85% of OTA, whereas average in vitro adsorption abilities of organic (30.52 ± 3.50%) and mixed (32.00 ± 2.60%) adsorbents were significantly lower.

4. In the investigation of absorption in everted sacs of broiler duodenal segments (Everted Duodenal Sacs Procedure), higher OTA adsorption in gut was exhibited by organic adsorbent, 74.26 ± 4.48%. Furthermore, the mean adsorption efficiency of mixed and inorganic adsorbent was 65.26 ± 4.76% and 45.75 ± 7.14%, respectively.

5. In the in vivo investigation, broilers were fed for 21 d on diets containing 2 mg/kg of OTA and supplemented with inorganic (Mz), organic (Ms) or mixed adsorbent (Mf) at the recommended concentration of 2 g/kg of feed. All three adsorbents significantly decreased OTA residue concentrations in the pectoral muscle and livers, but the order of effectiveness was mixed > organic > inorganic. The most efficient was the mixed adsorbent which decreased residue concentration by 72.50% in pectoral muscle and 94.47% in livers.

6. The Everted Duodenal Sac in vitro method provided results similar to those obtained in the in vivo study. However, further studies are required to investigate the efficiencies of adsorbents against various mycotoxins using this method.     

Influence of Clostridium perfringens and its toxin in germ-free chickens

Twenty-one of 56 germ-free chickens died after receiving an oral inoculation of a broth culture of Clostridium perfringens. At necropsy there was detachment and disruption of the epithelial layer at the tips of villi and sloughed epithelial cells in the duodenum. These are characteristic lesions of necrotic enteritis. Germ-free chickens receiving either purified alpha-toxin or the supernatant of broth cultures of C perfringens died, but no bird died after receiving supernatant of broth cultures neutralised by anti-alpha-toxin serum. It was concluded that alpha-toxin of C perfringens was the cause of death in young germ-free chickens.     

The effect of hen-egg antibodies on Clostridium perfringens colonization in the gastrointestinal tract of broiler chickens

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n = 10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n = 15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.     

Changes in the caecal microflora of chickens following Clostridium perfringens challenge to induce necrotic enteritis

Necrotic enteritis is a disease of considerable economic importance to the global poultry industry. Clostridium perfringens has long been recognised as the etiological agent of the disease. However, disease initiation and progression is complex and appears to be precipitated by a range of predisposing factors. The present study investigated microbial interactions in the caecum of birds challenged with C. perfringens that developed necrotic enteritis. Bacterial populations of healthy and diseased birds, across two independent animal trials, were characterised by pyrosequencing of the V1-V3 region of 16S rRNA genes. Significant changes in the microbiota of infected birds were detected. Most of the affected bacterial species, including a number of butyrate producers, were reduced in abundance in infected birds compared to uninfected controls and a number of phylotypes, classified as Weissella species, were also more abundant in healthy birds. Conversely, some bacterial groups were more abundant in the C. perfringens-infected birds, for example, members of an unclassified order of Mollicutes showed a 3.7-fold increase in abundance in infected birds. Representative sequences from this novel order shared 99% identity with sequences previously detected in intestinal microbiota of chickens and humans, and have previously been shown to be represented in a number of samples originating from irritable bowel syndrome disease patients. We speculate that these newly identified perturbations in the composition of caecal microflora may play a role in the development and manifestation of necrotic enteritis.     

Incidence of Clostridium perfringens in broiler chickens and their environment during production and processing.

During a calendar year, a study was conducted involving 16 broiler flocks on four different farms, two farms belonging to each of two major U.S. poultry integrators. As determined by the detection of Clostridium perfringens in fecal or cecal samples, 15 (94%) of the flocks became positive for this bacterial enteropathogen, and only one remained negative throughout the 6-to-8-wk rearing period. Paper pads beneath chicks that were transported from the hatchery to the rearing house were contaminated with C. perfringens in 15 (94%) of the flocks. When sampled biweekly through grow out, 13 of the flocks were C. perfringens positive at 2 wk of age. These results suggest that colonization of the intestinal tract of broilers by C. perfringens is an early event. Of the environmental samples, all but feed in the hopper were contaminated before placement for at least one of the rearing periods. All sample types were contaminated at some point during the 6-to-8-wk grow-out period. Of the on-farm environmental samples, the highest incidences (percentage positive) of C. perfringens were detected in wall swabs (53%), fan swabs (46%), fly strips (43%), dirt outside the house entrance (43%), and swabs of workers' boots (29%). Birds were usually transported to the processing plant in coops that were already contaminated with C. perfringens. In the plant, C. perfringens was isolated more frequently from samples of scald water than from those of chill water. Clostridium perfringens was recovered from broiler carcasses after chilling in 13 (81%) of the 16 flocks. The proportion of C. perfringens-positive carcasses for the contaminated flocks ranged from 8% to 68% with a mean of 30%.     

In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens

1. Three bacterial phytases derived from Bacillus, Escherichia coli or Klebsiella were compared with a phytase derived from Aspergillus niger in vitro and in vivo. 2. The in vitro results indicated that Aspergillus, E. coli and Klebsiella phytase displayed their activity optima in an acid pH range while Bacillus phytase did so in neutral pH. 3. The trials also revealed that only Bacillus phytase is more resistant to heat treatments, while E. coli and Klebsiella phytases are more stable against proteolytic inactivation. 4. In vivo phytases derived from Aspergillus, Bacillus, E. coli, Klebsiella or a combination of Bacillus and E. coli improved the utilisation of phosphorus (P balance) significantly to 0.54, 0.54, 0.55, 0.55 or 0.58, respectively, compared to 0.42 in the negative control. 5. The phytases used in this study seemed to be equally effective in improving P utilisation regardless of proposed intestinal site of activity. Combination of phytases acting in the gizzard with phytases acting in the intestine seems to be a promising way to further improving in vivo efficacy of phytases in poultry.     

Molecular detection and characterization of Clostridium perfringens toxin genes causing necrotic enteritis in broiler chickens

Tropical Animal Health and Production - A total of 464 samples comprising of cloacal swabs from necrotic enteritis suspected live birds (191), intestinal scrapings from dead birds with symptoms of...     

Optimized necrotic enteritis model producing clinical and subclinical infection of Clostridium perfringens in broiler chickens

In this study we assessed the roles of Eimeria infection and dietary manipulation (feeding a diet with a high level of fishmeal) in an Australian necrotic enteritis (NE) challenge model in broiler chickens. An experiment was designed to test the hypothesis that Eimeria infection and dietary manipulation, i.e., inclusion of fishmeal in the diet, are necessary to induce NE experimentally. The results showed that the combination of Eimeria administration and fishmeal feeding had a significant effect on induction of clinical and subclinical Clostridium perfringens infection. The majority of the mortality that occurred during the second week of the trial was due to an NE outbreak following the C. perfringens challenge. The mortality rate of the birds was 12.00% for the high-fishmeal (HFM; 500 g/kg) group and 9.33% for the low-fishmeal (LFM; 250 g/kg) group when the birds were subjected to C. perfringens and Eimeria. Fishmeal alone did not induce significant mortality in birds challenged only with C. perfringens but showed a significantly higher C. perfringens count than the non-fishmeal (NFM) control group. Eimeria administration had a significant effect on NE-related mortality but did not have an effect on the C. perfringens count. In accordance with the time course of bird mortality, it can be determined that of the 3 successive days of oral gavage with C. perfringens, the first inoculation was essential for inducing NE, but the third had no additional effect on NE-related mortality. Also, reducing the fishmeal level from 500 to 250 g/kg had no negative impact on the reproducibility of the model. It may be concluded that NE can be consistently induced under experimental conditions by feeding broilers a diet containing 250 g/kg fishmeal, using a single inoculation with low numbers of Eimeria, administering one or two oral C. perfringens inoculations, and maintaining appropriate ambient temperatures and diets.     

Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.     

不同地区鸡源产气荚膜梭菌的毒素型鉴定和药敏试验

为探明我国鸡群中产气荚膜梭菌(Clostridium perfringens,Cp)不同毒素型分布特征及携带主要致病毒素基因和对饲用抗菌素的抗药谱,于2013年7月至2014年5月间从四川等7省17个肉鸡生产较为集中的地区采样692份,分离鉴定获得Cp菌株214株,分离率31%。经多重PCR方法鉴定,其中有206株为A型Cp,8株为C型。利用特异性单一PCR方法检测发现:有87株Cp携带β2毒素基因,阳性率为41%;5株携带Net B毒素基因,阳性率为2.3%。以最小抑菌浓度法(MIC)检测的常见饲用抗菌素敏感性结果显示:那西肽、弗吉尼亚霉素和效霉素有广泛的抑菌作用,四川德阳(2013)、四川眉山(2013)和广东鹤山(2013)的分离菌株对所测试的11种药物均表现敏感,而广西桂林(2014)的菌株仅对那西肽敏感。结果表明我国鸡源Cp具有明显的毒素型、主要致病毒素基因携带状况和抗药谱的地区差异。     

Enterotoxigenic Clostridium perfringens type A isolated from intestinal contents of cattle, sheep and chickens.

One hundred and fourteen strains of Clostridium perfringens, isolated from the intestinal contents of cattle, sheep, and chickens with enteritis or other disease conditions were studied for their ability to produce enterotoxin. Reversed passive hemagglutination, fluorescent antibody and immunodiffusion tests were used. On the basis of the reversed passive hemagglutination titres, supported by the other two tests, enterotoxigenicity of the strains was arbitrarily classified into two categories: highly enterotoxigenic and potentially enterotoxigenic, with 12% falling into each category. All the highly enterotoxigenic strains originated from cases of enteritis and included all three animal species. Apart from enterotoxigenicity, one C. perfringens strain produced beta toxin (type C) and 21 strains produced large amounts of alpha-toxin. The latter strains were predominantly associated with necrotic enteritis in chickens.     

Effect of a synbiotic on the intestinal microflora of chickens

The current experiment was conducted to evaluate the effect of a synbiotic (Biomin Imbo) on intestinal microflora of Ross broiler chickens. A total of 200 male chickens were randomly divided into 20 groups of 10 birds each, kept in 20 pens (1.5 × 1 m each), and fed for 42 d at different synbiotic levels. Treatments included (1) a basal diet without synbiotics (control), (2) a basal diet with synbiotic levels proposed by the manufacturer, (3) a basal diet with synbiotic levels 25% lower than those proposed by the manufacturer, (4) a basal diet with synbiotic levels 50% higher than those proposed by the manufacturer, and (5) a basal diet with synbiotic levels 25% higher than those proposed by the manufacturer. At the end of the 42-d feeding period, 1 bird was randomly selected from each experimental unit, humanely euthanized, and the cecum was removed to measure the microbial population. The cecal contents were collected in discharge containers for microbial cultures, and counts were conducted after microbial culture. The addition of the synbiotic reduced Escherichia coli and total coliform populations in the intestines of broiler chickens. Conversely, different levels of probiotic increased the numbers of lactobacilli in the intestine of broiler chickens. Concentrations of the synbiotic higher than the recommended levels in the diet increased the lactic acid bacteria population in the gut of broiler chickens.     

Influence of dietary mushroom Agaricus bisporus on intestinal morphology and microflora composition in broiler chickens

In this study, we evaluated the intestinal morphology and bacteria populations in broiler chickens fed for six weeks diets that contained different amount of the mushroom Agaricus bisporus. Ninety day-old female chicks were randomly divided into three dietary treatments, each with three replicates kept in floor pens and fed a basal diet supplemented with the dried mushroom at levels of 0, 10 or 20 g/kg fresh feed. Feed and water were offered to birds ad libitum. The morphological examinations of the intestine were carried out on 1-cm long excised segments from duodenum, jejunum and ileum. The populations of total aerobes, total anaerobes, Lactobacilli spp., Bifidobacteria spp., Escherichiacoli, Bacteroides spp. and Enterococci were enumerated in ileum and caecum by conventional microbiological techniques using selective agar media. The results of the study showed that dietary mushroom supplementation did not significantly affect intestinal morphology at either level of inclusion. Morphometrical parameters of depth of duodenum, jejunum and ileum crypt and height of villi revealed no differences amongst dietary treatments. In the ileum, Lactobacilli spp. were higher in birds supplemented at the level of 20 g/kg compared to controls; however, other measurements of bacteria loads were similar amongst the three dietary treatments. In the caecum, Lactobacilli spp. and Bifidobacteria spp. loads were higher in birds supplemented at either level of inclusion compared to control birds, although these did not differ between the two levels of supplementation. In conclusion, dietary mushroom supplementation may beneficially affect intestinal health of broiler chickens.