Role of Coccidia in the occurrence of necrotic enteritis of chickens

Clostridium perfringens type A, Eimeria acervulina, and Eimeria necatrix were used to produce necrotic enteritis in chickens. The disease was produced in all groups of birds that received feed contaminated with C. perfringens. Mortality due to necrotic enteritis was highest (53%) in birds infected with E. acervulina before infection with clostridia. There was a significant difference in mortality rates between birds infected with E. acervulina and birds infected with E. necatrix before infection with C. perfringens. Mortality rates also differed significantly between the group infected with E. necatrix and the group that received only feed contaminated with C. perfringens. It was concluded that under field conditions, coccidia can play a significant role in the occurrence of necrotic enteritis when a sufficient number of toxigenic strain of C. perfringens type A is present. The pathological changes induced by clostridia and coccidia are described.     

In vitro activity of chloropolysporin-C, a new glycopeptide-group antibiotic, on Clostridium perfringens isolates and its in vivo activity against C perfringens and other intestinal microflora of the caeca of broiler chickens

The influence of chloropolysporin-C, a new glycopeptide antibiotic, on in vitro activity of Clostridium perfringens isolates and its effects on the intestinal microflora of broiler chickens was examined. The in vitro sensitivity of 88 isolates of C perfringens to four antimicrobial agents, including chloropolysporin-C, was tested by an agar dilution method. The antibiotics used all had minimum inhibitory concentration levels of 6.25 micrograms ml-1 or less against this organism. Changes were examined in the intestinal microflora of broiler chickens fed a diet containing chloropolysporin-C to obtain basic data on the mechanisms by which the antibiotic aided livestock production. No clinical findings were recognised in chickens tested during the period of antibiotic administration. A decrease in viable cells of the clostridia was the principal response recognised during the period of drug administration in feed. Among the other microflora, chloropolysporin-C led to a significant response among Gram-positive bacteria, but no changes in the total bacterial count.     

小麦日粮非淀粉多糖和木聚糖酶对肉仔鸡肠道微生物区系的影响

采用传统微生物培养和PCR-DGGE 2种方法，针对肠道内几种微生物的数量和优势菌的多样性进行研究。将90只AA肉仔鸡随机分成3组，分别饲喂玉米-豆粕型，小麦-豆粕型和小麦-豆粕型添加木聚糖酶3种日粮。结果表明，小麦非淀粉多糖有增加肠道内几种微生物数量的趋势，其中显著增加了回肠和盲肠厌氧菌总数以及盲肠粪链球菌数量（P＜0．05），而PCR-DGGE结果表明，小麦NSP改变了肠道内微生物的种群结构，降低了种群的数量（P＜0．05）。添加木聚糖酶对回肠和盲肠的微生物数量以及种群数量有影响，但未表现出显著性差异（P＞0．05）。说明小麦非淀粉多糖作为微生物发酵的底物对肠道厌氧微生物产生了增殖效应，但却抑制了其他微生物的生长，导致肠道内微生物结构发生改变。     

Determination of Ileum Microbial Diversity of Broilers Fed Triticale- or Corn-Based Diets and Colonized by Salmonella

Diversity of the bacterial communities in the ileum of broilers was characterized using denaturing gradient gel electrophoresis. Denaturing gradient gel electrophoresis separation of polymerase chain reaction amplicons of the V2–V3 variable regions of the 16S rDNA is a common method to profile community diversity and has been used to assess the effects of diet and antibiotics on the ileal bacterial community of chickens. Broilers raised either on litter floor or in cage batteries were fed either a finely ground corn- (control), a finely ground triticale-, or a whole triticale-based diet from 0 to 42 d. Microbial DNA was extracted from the ileum content of 42-d-old broilers, and the 16S rDNA gene was amplified by polymerase chain reaction and the amplicons separated by denaturing gradient gel electrophoresis. Diversity indexes including richness, evenness, diversity, and pairwise similarity coefficients were calculated. Diversity indexes were related to the dietary treatments, housing designs, and to changes in Salmonella colonization of broiler ceca as characterized by the most probable number method. Higher microbial diversity indexes were observed among birds fed whole triticale-based diets and reared on litter floors. In contrast, finely ground grain treatments had lower diversity and higher Salmonella prevalence than the whole triticale treatment. The data indicated that combination of high dietary fiber content and increased coarseness of the diet by feeding whole triticale stimulated microbial community diversity and discouraged Salmonella colonization, perhaps through a competitive exclusion-type mechanism.     

Reciprocal competitive exclusion of salmonella and Escherichia coli by native intestinal microflora of the chicken and turkey

Both the native intestinal microflora of chickens that protected chicks against salmonellae and Escherichia coli and native turkey intestinal microflora were evaluated for their reciprocal protective capacity in both species against Salmonella typhimurium and a pathogenic strain of E. coli. Nalidixic-acid-resistant forms of the S. typhimurium and E. coli strains were used in seeder-bird and individual-bird challenge tests. Reciprocal protection was provided by native chicken and turkey intestinal microflora in chicks and poults against S. typhimurium and the pathogenic strain of E. coli. The chicken and turkey microflora appeared to be equally effective in protecting the two species from S. typhimurium, but protection against E. coli was somewhat greater in the chicken than in the turkey.     

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.     

Assessment of the qualitative variation in bacterial microflora among compartments of the intestinal tract of dogs by use of a molecular fingerprinting technique

OBJECTIVE: To evaluate the qualitative variation in bacterial microflora among compartments of the intestinal tract of dogs by use of a molecular fingerprinting technique. ANIMALS: 14 dogs (similarly housed and fed identical diets). PROCEDURE: Samples of intestinal contents were collected from the duodenum, jejunum, ileum, colon, and rectum of each dog. Bacterial DNA was extracted from the samples, and the variable V6 to V8 region of 16S ribosomal DNA (gene coding for 16S ribosomal RNA) was amplified by use of universal bacterial primers; polymerase chain reaction amplicons were separated via denaturing gradient gel electrophoresis (DGGE). Similarity indices of DGGE banding patterns were used to assess variation in the bacterial microflora among different compartments of the intestine within and among dogs. Bacterial diversity was assessed by calculating the Simpson diversity index, the Shannon-Weaver diversity index, and evenness. RESULTS: DGGE profiles indicated marked differences in bacterial composition of intestinal compartments among dogs (range of similarity, 25.6% to 36.6%) and considerable variation among compartments within individual dogs (range of similarity, 36.7% to 579%). Similarities between neighboring intestinal compartments were significantly greater than those between non-neighboring compartments. Diversity indices for the colon and rectum were significantly higher than those of the duodenum, jejunum, and ileum. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the different intestinal compartments of individual dogs appear to host different bacterial populations, and these compartmental populations vary among dogs. In dogs, fecal sample analysis may not yield accurate information regarding the composition of bacterial populations in compartments of the gastrointestinal tract.     

Effects of necrotic enteritis challenge on intestinal micro-architecture and mucin profile

1. This study investigated the effect of Eimeria spp./Clostridium perfringens induced necrotic enteritis and traditional antibiotic preventatives on intestinal micro-architecture and mucin profile. 2. A total of 600 Cobb 500 broiler chickens were randomly assigned to the following three groups: (i) unchallenged, (ii) challenged, and (iii) zinc bacitracin/monensin (ZnB/monensin) (n = 25 chickens/pen, 8 pens/group). The challenged and ZnB/monensin chickens were individually inoculated with Eimeria acervulina, E. maxima and E. tenella and C. perfringens type A (EHE-NE18) at 9 and 15 d post-hatch respectively, to induce necrotic enteritis. 3. The challenge procedure significantly decreased villus height, increased villus width and increased crypt depth in the challenged compared to the unchallenged chickens. Zinc bacitracin and monensin maintained villus-crypt structure similar to that of the unchallenged chickens. 4. Mucin profile was not affected by Eimeria spp./C. perfringens challenge as demonstrated by periodic acid-Schiff and high iron diamine-alcian blue pH 2 x 5 staining. Zinc bacitracin and monensin decreased the number of intestinal mucin-containing goblet cells. 5. Lectin histochemistry showed a trend towards greater Arachis hypogea (PNA) reactivity in unchallenged chickens. 6. In summary, Eimeria spp./C. perfringens challenge disrupted intestinal micro-architecture; however, challenge did not appear to affect intestinal mucin profile. Traditional antibiotics, zinc bacitracin and monensin maintained micro-architecture.     

Cecal bacterial communities in wild Japanese rock ptarmigans and captive Svalbard rock ptarmigans

Preservation of indigenous gastrointestinal microbiota is deemed to be critical for successful captive breeding of endangered wild animals, yet its biology is poorly understood. Here, we investigated cecal bacterial communities in wild Japanese rock ptarmigans (Lagopus muta japonica) and compared them with those in Svalbard rock ptarmigans (L. m. hyperborea) in captivity. Ultra-deep sequencing of 16S rRNA gene indicated that the community structure of cecal microbiota in wild rock ptarmigans was remarkably different from that in captive Svalbard rock ptarmigans. Fundamental differences between bacterial communities in the two groups of birds were detected at the phylum level. Firmicutes, Actinobacteria, Bacteroidetes and Synergistetes were the major phyla detected in wild Japanese rock ptarmigans, whereas Firmicutes alone occupied more than 80% of abundance in captive Svalbard rock ptarmigans. Furthermore, unclassified genera of Coriobacteriaceae, Synergistaceae, Bacteroidaceae, Actinomycetaceae, Veillonellaceae and Clostridiales were the major taxa detected in wild individuals, whereas in zoo-reared birds, major genera were Ruminococcus, Blautia, Faecalibacterium and Akkermansia. Zoo-reared birds seemed to lack almost all rock ptarmigan-specific bacteria in their intestine, which may explain the relatively high rate of pathogenic infections affecting them. We show evidence that preservation and reconstitution of indigenous cecal microflora are critical for successful ex situ conservation and future re-introduction plan for the Japanese rock ptarmigan.     

The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens

1. The influence of feed grinding (coarsely or finely ground feed) and feed form (mash or pellets) on the intestinal environment was investigated in a growth experiment with broiler chickens taking the intestinal microflora, intestinal viscosity, and the activities of pancreatic digestive enzymes into consideration. 2. As compared to mash the feeding of pellets was associated with a significantly higher body weight due to increased feed intake and improved feed utilisation. 3. Pellet-fed birds had significantly decreased gizzard weights, a higher gizzard pH and a lower intestinal pH than mash-fed birds. 4. Pellet-fed birds had significantly lower relative pancreas weights and lower activities of pancreatic digestive enzymes (amylase, lipase, chymotrypsin), which indicates the existence of a feedback mechanism, which may have been triggered by the intestinal concentration of enzymatically hydrolysed products or of the respective digestive enzymes. 5. Pellet-fed birds had larger numbers of coliform bacteria and enterococci in the ileum and a reduced number of Clostridium perfringens and lactobacilli in the distal end of the digestive tract (caeca and rectum). Microbial fermentation in terms of volatile fatty acid (VFA) concentration was found to be significantly higher in the caeca of pellet-fed birds than in mash-fed birds.     

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.     

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.     

Fumonisins affect the intestinal microbial homeostasis in broiler chickens,predisposing to necrotic enteritis

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 ± 0.016) compared to the control (0.14 ± 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 ± 0.30 versus 6.3 ± 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 ± 2.22% versus 29.8 ± 5.46%).

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0234-8) contains supplementary material, which is available to authorized users.     

Predisposing factors and prevention of Clostridium perfringens-associated enteritis

Clostridium perfringens is one of the major causes of intestinal disease in humans and animals. Its pathogenicity is contributed to by the production of a variety of toxins. In addition, predisposing environmental factors are important for the induction of C. perfringens-associated enteritis as shown by infection models. Environmental contamination, gastric and intestinal pH, intestinal microflora, nutrition, concurrent infections, and medical interventions may influence the intestinal colonization, growth, and toxin production by C. perfringens. Prevention of C. perfringens-associated enteritis may be mediated by the use of feed additives like probiotics, prebiotics, organic acids, essential oils, bacteriophages, lysozymes, bacteriocins, and antimicrobial peptides. Here we summarize and discuss published data on the influence of different environmental predisposing factors and preventive measures. Further research should focus on feed composition and feed additives in order to prevent C. perfringens-associated enteritis.     

Induction of maggot antimicrobial peptides and treatment effect inSalmonella pullorum-infected chickens

Antimicrobial peptides (AMP) are important components of the host innate immune response, as they exert broad-spectrum antimicrobial activities against pathogenic microbes. The AMP allow housefly larva (maggots) to live in harsh environments filled with pathogenic bacteria. In this study, maggot AMP were induced by incubation with inactivatedSalmonella pullorum and crudely extracted. The concentration and antimicrobial activity of the maggot AMP were then measured. In bird experiments, chickens were artificially infected withS. pullorum, and the maggot AMP extracts were used to treat the infected chickens. The expression level of AMP was significantly enhanced byS. pullorum stimulation, and the antibiotic activity of theS. pullorum-induced AMP was significantly stronger than that of the noninduced AMP, especially againstS. pullorum. In the bird experiments, based on survival rate, blood indicators, and intestinal bacterial changes, maggot AMP and antibiotics were successful in treating theS. pullorum-infected chickens. In conclusion, AMP have the potential for further development as a convenient, alternative antibiotic strategy to reduce the use of antibiotics and disease resistance.     

Effect of avilamycin, tylosin and ionophore anticoccidials on Clostridium perfringens enterotoxaemia in chickens

In order to study the prophylactic and metaphylactic effect of antomicrobial growth promoters and ionophorous anticoccidials on the incidence of Cl. perfringens enterotoxaemia in chickens, experimental attempts were performed with 675 chickens in 27 trials. The birds were intraduodenally infected with Cl. perfringens type A (ATCC 3624). The following antimicrobial growth promoters and ionophore anticoccidials were used either on their own or in combination: avilamycin, narasin, monensin and tylosin. While infected and non-medicated trials showed an average incubation period of 1 week, clinical symptoms occurred 2-4 days later in infected and medicated birds. Avilamycin medicated birds had the longest incubation period. In the infected and non-medicated trials, a mortality rate of 16%-36% was noted within 3 weeks post infection. The avilamycin trials showed a mortality rate of 0-8% (0-2 birds died) and the narasin and monensin a mortality rate of 0-8%, respectively. In the combination groups (monensin + avilamycin or narasin + avilamycin), the mortality rate ranged from 0 to 4%. Tylosin showed a very good metaphylactic/therapeutic effect against Cl. perfringens enterotoxaemia. Following infection, medicated birds showed a significantly better bodyweight gain than the chickens, whose feeds had not been supplemented. From epidemiological point of view, the systematic prevention of coccidiosis is a key in the control of Cl. perfringens enterotoxaemia in chickens.     

Necrotic Enteritis in Broiler Chickens II. Pathology and Proposed Pathogenesis

The intestines from 124 dead, sick and normal broiler chickens from 24 cases of necrotic enteritis were subjected to histological examination. Tissue sections from the duodenum, jejunum, ileum and ceca from each broiler were examined histologically for lesions of necrotic enteritis and the presence of coccidia. Lesions of necrotic enteritis were present in one or more areas of the intestine in all but six of 94 dead or sick birds and they were most common and severe in the jejunum. Coccidia were found in only small numbers in both diseased and normal birds.

Brown and Brenn stained sections showed Gram-positive bacilli intimately associated with early necrotic lesions on the tips of villi. Tissue sections from the intestines of sick birds permitted a proposed pathogenesis for this disease with the lesion starting at the tips of villi.

The similarity in pathogenesis and pathological lesions in this disease of broilers and Clostridium perfringens type C enteritis in baby pigs is discussed.

     

Entérite nécrotique chez le pollet de gril III. Etude des facteurs favorisant la multiplication de Clostridium perfringens et la transmission expérimentale de la maladie

Necrotic enteritis was reproduced experimentally in two week old broiler chickens by intravenous injection and also by oral administration of a pure culture of Clostridium perfringens. In the first experiment, gross and microscopic intestinal lesions, typical of necrotic enteritis, were observed in all diseased birds and mortality was obtained only in the group of birds that were injected with 0.4 ml or more of the pure culture of the microorganism. In the second experiment, the highest mortality was noted in the group of birds that received orally, in addition to the Clostridium culture, a solution of sodium bicarbonate, to obtain an alkaline intestinal content and opium to decrease the intestinal peristaltism. The gross and microscopie intestinal lesions of the diseased and killed birds were more severe than those observed in the other groups and were similar to those encountered in field outbreaks of necrotic enteritis.     

The influence of whole wheat feeding on Salmonella infection and gut flora composition in broilers

In an experiment with broilers infected with a rifampicin-resistant Salmonella Typhimurium strain at 15 days of age, it was demonstrated that whole wheat feeding influenced the course of infection. The pH in the contents of the gizzard decreased as the amount of whole wheat in the diet increased over time. Following infection, lower numbers of Salmonella Typhimurium were found in the gizzard and ileum of birds receiving whole wheat compared to pellet-fed birds. However, there was no difference with respect to the numbers of Salmonella Typhimurium in the ceca and rectum between the two experimental groups. Quantitative cultivation of different groups of bacteria belonging to the natural intestinal microflora showed that whole wheat feeding significantly reduced the numbers of Clostridium perfringens in the intestinal tract of the birds. The present results indicate that the gizzard has an important function as a barrier organ, one that prevents pathogenic bacteria from entering the distal digestive tract.     

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.