Experimental reproduction of necrotic enteritis in the chicken. 2. Further mono- and polyinfections with CL. perfringens and coccidia with special reference to ground-kept chickens]

Coccidial oocysts and/or vegetative germs, spores, and toxins of Cl. perfringens Type A were used in mono-infection and poly-infection experiments on SPF chicken aged seven and 56 days and kept under different conditions. Necrotic enteritis was regularly reproduced in all experimental groups with polyinfections. In chicken aged seven days necrotic enteritis was reproduced even after repeated mono-infections with 4 X 10(9) gerus and toxin of Cl. perfringens. The loss figures recorded from ground-kept infected groups were higher than those established from the cage-kept animals. The pathologica-anatomic findings recorded from the dead chicken included necrotic, ulcerative, and catarrhal to haemorrhagic intestinal inflammations, with necrotic enteritis being most strongly pronounced in the ileum and jejunum. All infected groups lost not only animals but weight as well. Their average weight on the 21 st day from the onset of infection was up to 26.4 per cent lower than that of the control groups.     

Toxinotypes of Clostridium perfringens isolated from sick and healthy avian species.

Currently, the factors/toxins responsible for Clostridium perfringens-associated avian enteritis are not well understood. To assess whether specific C. perfringens' toxinotypes are associated with avian enteritis, the isolates of C. perfringens from 31 cases of avian necrotic or ulcerative enteritis submitted between 1997 and 2005 were selected for retrospective analysis using multiplex PCR. C. perfringens was isolated from chickens, turkeys, quail, and psittacines. The toxinotypes of isolates from diseased birds were compared against the toxinotype of 19 C. perfringens isolates from avian cases with no evidence of clostridial enteritis. All C. perfringens isolates were classified as type A regardless of species or disease history. Although many isolates (from all avian groups) had the gene encoding the C. perfirngens beta2 toxin, only 54% produced the toxin in vitro when measured using Western blot analysis. Surprisingly, a large number of healthy birds (90%) carried CPB2-producing isolates, whereas over half of the cpb2-positive isolates from diseased birds failed to produce CPB2. These data from this investigation do not suggest a causal relationship between beta2 toxin and necrotic enteritis in birds.     

Necrotic enteritis potential in a model system using Clostridium perfringens isolated from field outbreaks

Necrotic enteritis is an enteric disease of avian species caused by the anaerobic bacterium Clostridium perfringens. The disease is regularly controlled in the broiler chicken industry with antimicrobials in feed but is reemerging in areas such as Europe where there is a ban on antimicrobials as growth promoters. To study prospective therapies, researchers must be able to reproduce this disease in a controlled environment, but this is not always possible because of differences in the pathogenicity of C. perfringens strains. Our objective was to test the potential of five isolates (SNECP43, 44, 47, 49, and 50), taken from field cases of necrotic enteritis, at recreating the disease in a controlled challenge experiment. SNECP43 and 50 were derived from a common clone, with SNECP50 passed in vivo and SNECP43 subcultured in vitro. Four hundred birds were divided into 16 pens, with three pens each receiving one of five treatments, with one control pen. Day-old birds were raised on a high wheat-based diet to promote necrotic enteritis development and were challenged with between 3.4 x 10(9) and 3.2 x 10(11) colony-forming units (cfu) of C. perfringens in feed for a period of 24 hr starting on day 13 of the challenge experiment. Lesion scores were assessed on two birds per pen sacrificed on day 17 and on any dead birds during the 25-day study. Growth performance was assessed up to 25 days, and mortality recorded throughout. Only SNECP50 produced necrotic enteritis mortalities significantly different (P < or = 0.05) from the control. The five isolates were also typed using pulsed-field gel electrophoresis to assess their genetic relatedness. All epidemiologically unrelated isolates were deemed genetically unrelated, whereas SNECP43 and 50 differed by only a single minor band. Toxin type was assessed using polymerase chain reaction (PCR), which was also used for the detection of the gene encoding the beta2-toxin.     

Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm

Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study.     

Effects of Eimeria brunetti infection and dietary zinc on experimental induction of necrotic enteritis in broiler chickens.

Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis. Inoculation with sporulated E. brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens. Feed was supplemented with zinc at 1000 ppm. Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc. Coccidial lesion scores were also highest in that group. Birds infected with E. brunetti and C. perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E. brunetti only, regardless of zinc supplementation. Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation. Zinc was tested for effects of alpha toxin production in vitro. In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin. Addition of zinc partly protected the toxin from the action of trypsin.     

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.     

Etiology and pathogenesis of necrotic enteritis

Sporulated oocysts of Eimeria acervulina were administered orally to cage-housed broilers at a dose of 3.5 X 10(5) resulted in mild subclinical coccidiosis. Clostridium perfringens incorporated in feed at a level of 2.5 X 10(8) organisms/g. produced lesions characteristic of necrotic enteritis. Mortality of 8% (7/80) occurred in birds fed a ration inoculated with Cl. perfringens alone. Mortality of 35% (28/80) was observed in birds which received an oral dose of E. acervulina and which were fed simultaneously with a ration containing Cl. perfringens. Birds which were fed an inoculated ration two days after an oral dose of E. acervulina showed 41% (33/80) mortality. Birds which received an inoculated ration for two days before administration of an oral dose of E. acervulina demonstrated 18% mortality (15/80). Birds which were fed an inoculated ration four days after an oral dose of E. acervulina showed 10% mortality. Infection with E. acervulina reduced the pH of intestinal contents with a simultaneous depression in serum protein. A 39% increase in intestinal passage time from 178 to 248 minutes occurred on the fifth day after infection with E. acervulina. These experiments suggest that necrotic enteritis, attributed to proliferation of a toxigenic strain of Cl. perfringens, followed intestinal stasis and minimal lesions induced by mild intestinal coccidiosis.     

Necrotic enteritis in turkeys.

Three flocks of turkey hens (16,000 each) between 7 and 12 weeks of age experienced outbreaks of necrotic enteritis. Necropsy revealed a dilated duodenum and jejunum with mucosal surfaces covered with a diphtheritic membrane. Intestinal scrapings showed very few oocysts of Eimeria sp. Histopathological findings were compatible with necrotic enteritis but with deeper, more severe lesions than in broiler chickens. Clostridium perfringens was isolated by anaerobic culture from the intestinal contents. Mortality returned to normal after ampicillin or tetracycline was added to the drinking water.     

Reproduction and treatment of necrotic enteritis in broilers.

A change in ration from one containing 50% fish meal to a standard chick starter containing 10(8) Clostridium perfringens/g of feed resulted in the production of necrotic enteritis in 2-week-old broiler chicks. Lincomycin added to the inoculated ration at a concentration of 20 g/907.2 kg significantly reduced mortality in the chicks. Inoculation of broth cultures of C perfringens directly into the duodenum, using a surgically inserted tetrafluoroethylene resin tube, indicated a relationship existed between the number of C perfringens inoculated and the gross lesions. The disease could be consistently produced by this technique.     

Hemorrhagic enterocolitis and death in two felines (Panthera tigris altaica and Panthera leo) associated with Clostridium perfringens type A

Severe hemorrhagic enterocolitis was observed in a Siberian tiger (Panthera tigris altaica) and a lion (Panthera leo). Both animals developed acute depression, anorexia, and bloody diarrhea several days before death. Small and large intestines were diffusely congested, edematous, necrotic, and filled with hemorrhagic fluid, and mesenteric lymph nodes were enlarged and congested. Pure and abundant growth of gram-positive bacilli was obtained in culture under anaerobic conditions from the livers of both felines. Identification of highly virulent Clostridium perfringens Type A was based on pathologic lesions, hemolytic patterns, morphologic structure, and polymerase chain reaction. Animal inoculation assays indicated that C. perfringens Type A played an important role in the pathogenesis of both felines.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.

The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.

In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

产气荚膜梭菌菌落多重PCR方法的建立及初步应用

根据GenBank中已发布的产气荚膜梭菌α、β、ε、τ毒素基因序列,分别设计并合成针对4种毒素基因的特异引物,通过优化多重PCR反应条件,建立1种简单的产气荚膜梭菌定型菌落多重PCR方法。结果显示：A、B、C、D、E5型产气荚膜梭菌参考菌株均扩增出了相应的预期目的条带,而大肠杆菌、巴氏杆菌和芽孢杆菌则均未能扩增出相应条带;将单个菌落稀释100倍,仍能扩增出相应的目的片段,该方法对B型和E型参考菌株最低检测量分别为2．6×10^4cfu／mL、1．2×10^4cfu／mL。应用该多重PCR方法从106份样品中检测到30株产气荚膜梭菌且均为A型,其中病死鸡的盲肠内容物分离率为36．5％（19／52）,健康鸡群新鲜粪便样品分离率为20．4％（11／54）。本研究建立的多重PCR方法特异性强,敏感度高,重复性好,可以有效进行产气荚膜梭菌的快速检测及5种血清型的鉴别,对产气荚膜梭菌的感染及食品安全问题的研究均具有重要意义。     

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.     

Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation

Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining trays used to transport commercial chicks from the hatchery to the grow-out facility on the farm. These results suggest that C. perfringens contaminating the processed product could originate from facilities in the integrated poultry operation prior to grow out. In this study, samples were collected from the breeder farm, hatchery, previous grow-out flock, during grow out and after processing. In the first trial, C. perfringens was recovered from the breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, from processed carcasses, and from the breeder farm after processing in 4%, 30%, 4%, 0%, 2% and 16%, and 4% of the samples, respectively. In the second trial, the incidence of C. perfringens in samples collected from breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, and fromprocessed carcasses was 38%, 30%, 32%, 8%, 4%, and 8%, respectively. The genetic relatedness of the isolated strains as determined by ribotyping suggests that C. perfringens may be transmitted between facilities within the integrated broiler chicken operation.     

Emphysematous gastritis in a horse

A 12-year-old Morgan gelding was examined for colic of 3 days duration. Signs of depression, colic, diarrhea, and endotoxemia persisted despite aggressive medical therapy and surgical exploration. Culture results from gastric fluid and feces yielded many colonies of Clostridium perfringens. This organism also was recovered from peritoneal fluid 10 days after admission; consequently, the horse was euthanized. At necropsy, a localized gas-filled, necrotic stomach wall was found; many mucosal and submucosal gas blebs were visible. Culture of this tissue yielded Clostridium perfringens. Emphysematous gastritis is a fulminant infection of the stomach wall caused by gas-forming organisms that gain access to the submucosa via mucosal defects such as ulcers. This condition has been reported infrequently in people, and the case reported herein represents the first instance of emphysematous gastritis in the horse.     

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.     

Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status

Due to the diminished use of growth-promoting antibiotics in the European Union, Clostridium perfringens induced necrotic enteritis and subclinical disease have become important threats to poultry health. A study was set up to genotypically and phenotypically characterise C. perfringens isolates from poultry flocks with different health status. Animals from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of animals suffering from necrotic enteritis were analysed. A total of 27 isolates was obtained from 23 broiler flocks without clinical problems and 36 isolates were obtained from 8 flocks with clinical problems. Using PFGE typing, high genetic diversity was detected between isolates from different flocks. Isolates derived from flocks where disease outbreaks occurred were clonal within each flock, but each flock harboured a different clone. All isolates were of toxin type A. Isolates from 5 out of 35 PFGE types carried the cpb2 gene, encoding the beta2 toxin, and isolates from 2 out of 35 PFGE types harboured the cpe gene, encoding the enterotoxin. In vitro alpha toxin production for all isolates was quantified by enzyme-linked immunosorbent assay. It was shown that in vitro alpha toxin production of C. perfringens isolates from diseased flocks was not higher than in vitro alpha toxin production from isolates derived from healthy flocks.     

The influence of diet on necrotic enteritis in broiler chickens.

Necrotic enteritis was reproduced in broiler chickens by mixing cultures of Clostridium perfringens in the feed. Mortality due to necrotic enteritis was higher among chickens fed rations based on wheat, rye, barley, and oat groats than among chickens fed corn-based rations. Addition of pentosanase to a wheat-based diet did not affect the level of mortality due to necrotic enteritis. Addition of pectin and guar gum to different rations severely reduced growth rate and eliminated necrotic enteritis from test birds. Addition of glucose to a corn-based diet caused a small increase in mortality due to necrotic enteritis.     

Necrotic enteritis in broiler chickens. III. Reproduction of the disease.

Lesions typical of necrotic enteritis could be produced experimentally in from 11-26% of broiler chickens consuming feed containing approximately 10(7) Clostridium perfringens per gram. Highest mortality was produced using isolates from field cases of necrotic enteritis which were reisolated from experimental cases in the laboratory. Penicillin in the drinking water at 100,000 I.U./litre completely prevented mortality whereas chloramphenicol at 110 mg/litre delayed the onset and reduced the number of deaths compared to controls.     

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.