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.     

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.     

Cecal carriage of Clostridium perfringens in broiler chickens given Mucosal Starter Culture.

Day-of-hatch broiler chicks housed in isolation units were each given, by oral gavage, 0.1 ml of Mucosal Starter Culture (MSC) or saline control. Each of the treated and control chicks was subsequently given a composite culture of three strains of bacitracin-resistant Clostridium perfringens (Cp) previously isolated from chickens with symptoms of necrotic enteritis. Some chicks were maintained on a corn-based diet provided ad libitum. Others were given the feed supplemented with 50% rye (a predisposing factor for necrotic enteritis). At 7, 14, and 21 days after receiving Cp, chicks were euthanatized, and cecal contents were diluted and plated on selective agar containing bacitracin. For chicks on corn feed, Cp numbers were similar in control birds and birds given MSC in three of four trials. In two of the trials that demonstrated no effect of MSC on Cp numbers, enterotoxin presence was determined. The number of birds with detectable Cp enterotoxin in their small intestine and the mean toxin levels were lower in the MSC-treated birds. In a fourth trial with birds on corn-based feed, mean Cp numbers and the number of Cp-positive birds were lower in the MSC-treated birds. For the two trials involving chickens on rye-supplemented feed, Cp numbers and the percentage of Cp-positive birds were significantly reduced in MSC-treated birds compared with control birds. Enterotoxin in birds receiving the 50% rye diet was at low levels or not detected in control and MSC-treated birds. Results suggest that MSC may reduce intestinal proliferation of Cp, a causative agent of necrotic enteritis in poultry and of foodborne disease in humans.     

Enteritis as a cause of mortality in the western bluebird (Sialia mexicana)

Increased mortalities in adult western bluebirds utilizing nestboxes were noted in western Oregon during 1998 and 1999. A necrohemorrhagic enteritis was found in 8 of 10 birds submitted for necropsy. Acanthocephalan parasites were present in four of eight birds with enteritis. Microscopic changes consistent with necrotic or ulcerative enteritis were commonly present. Anaerobic culture of the intestine yielded Clostridium perfringens in three of three birds. Genotype analysis of two of these isolates revealed them to be C. perfringens type A. Bacterial enteritis is believed to be the cause of the increased mortality rate, but further investigation is required to prove a definitive link to a clostridial agent.     

Sandwich ELISA detection of Clostridium perfringens cells and alpha-toxin from field cases of necrotic enteritis of poultry

Sandwich ELISAs (sELISAs) for the detection of Clostridium perfringens cells and alpha-toxin were developed and used to screen intestinal samples from normal broiler chickens and from clinical cases of necrotic enteritis. The assays clearly distinguished between the two sets of samples. The sELISA absorbance values from samples obtained from the majority of healthy birds were low and those from the majority of necrotic enteritis cases were high. Together, the assays provide a suitable test for the rapid screening for the diagnosis of necrotic enteritis in poultry.     

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.     

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.     

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.     

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.     

Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens

The aim of this study was to analyse the genetic diversity among Clostridium perfringens isolates from Danish broiler chickens since both sick and presumably healthy animals were investigated. Isolates (n=279) collected from chickens from 25 farms were analysed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI. A high genetic diversity was found. Isolates with different PFGE types were toxin typed by PCR and all were found to be of type A. The results showed that healthy broiler chickens carried several different C. perfringens clones both within a flock and even within individual birds, whereas flocks suffering from necrotic enteritis (NE) or cholangio-hepatitis carried only one or two clones.     

Toxin production by Clostridium perfringens isolated from broiler chickens and capercaillies (Tetrao urogallus) with and without necrotizing enteritis.

A total of 192 isolates of Clostridium perfringens were isolated from 99 broiler chickens and 93 capercaillies (Tetrao urogallus). Fifty of the isolates from broilers and 44 of the isolates from capercaillies were from birds with necrotizing enteritis, and the remainder were from birds without this disease. The isolates were tested for the production of three major toxins (alpha, beta, and epsilon) and four minor toxins (theta, gelatinase, mu, and nu). All isolates were found to be C. perfringens type A. Alpha toxin was produced in significantly larger amounts by isolates from birds with necrotizing enteritis than by isolates from birds without the disease, regardless of bird species. Isolates from broilers produced significantly more alpha toxin than did isolates from capercaillies.     

An outbreak of necrotic enteritis in the ostrich farm in Korea

An acute disease with high mortality occurred in the ostrich farm and characterized by depression, severe diarrhea and sternal recumbency. Four dead ostriches of the farm were submitted to the National Veterinary Research & Quarantine Service, and diagnosed as necrotic enteritis. In the gross and histopathological examination, extensive diffuse fibrinonecrotic enteritis was found in the small intestine, especially jejunum. Clostridium perfringens was isolated from a pure culture from the duodenum and jejunum of these birds. Based on our current knowledge, this is the first report of an outbreak of necrotic enteritis in the ostrich in Korea.     

High mortality in egg layers as a result of necrotic enteritis

A new facility was designed to hold 1.8 million birds in 10 houses; chickens were placed in five of the houses, and the remaining five houses were under construction when this outbreak occurred. An increase in mortality was reported in five houses; however, mortality in house 7 was quite high. Well-fleshed birds were suddenly found dead without a significant drop in egg production. The middle and distal intestines were distended with gas, congested, thin walled, atonic, and bluish or pale in color with sloughed mucosa in some places. Necrotic enteritis was diagnosed as the cause of increased mortality. The ingesta in the crop occasionally contained flies. The 4-wk mortality in house 7 was 6.55% with a loss of 10,898 chickens. The 4-wk mortality rate in the other houses ranged from 0.54% to 1.98%. The houses affected with necrotic enteritis were treated for coccidiosis with amprolium because low numbers of the oocysts were present in the intestinal specimens of some of the chickens. Household bleach was added to the water at a dilution of one part bleach to 1040 parts water to control bacterial contamination. The fly (Musca domestica) population was out of control. Clostridium perfringens was isolated from the alcohol-washed macerated flies caught from houses 4 and 7. Dead flies were often seen in the feed troughs. The chickens may possibly have had C. perfringens infection as a result of consumption of dead flies or their secretions/excretions. The alcohol-washed, macerated, clarified fly extract from the affected houses caused death in 11 inoculated mice and paralysis in one mouse. Similarly, illness and mortality were present in four mice inoculated with clarified intestinal contents. The bacterium isolated on anaerobic culture was identified as C. perfringens by polymerase chain reaction. The disease was brought under control after straw was added and mixed in with the litter. As a result, the litter temperature increased, causing a decrease in the fly population. This study suggests that flies in the poultry houses acted as mechanical transmitters of C. perfringens and that the development of necrotic enteritis was by ingestion of bacteria present in the flies and their secretions/excretions.     

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.     

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.     

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.     

Ulcerative enteritis (quail disease) in lories

Ulcerative enteritis is found in a wide range of avian hosts but has not been described in psittacine birds. This case report describes ulcerative enteritis in four lories (two Trichoglossus sp. and two Eos sp.) that were found dead without any previous sign of disease. Macroscopically, all four birds showed good body condition. The only remarkable finding was a moderate dilatation of the small intestine with the presence of multiple yellow foci. Histologically, multiple ulcers extended into the submucosa and were filled with necrotic debris; bacteria and fibrin were observed in the intestinal mucosa. The liver and spleen exhibited a multifocal fibrinoid necrosis associated with a very moderate inflammatory reaction. Microbiological isolation revealed colonies of Clostridium colinum and Clostridium perfringens in the intestinal tract of the investigated birds.     

Reproducible infection model for Clostridium perfringens in broiler chickens

Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments were conducted, each involving four groups of chickens with each group kept in separate isolators. A coccidial vaccine given at 10 times the prescribed dosage was used to promote the development of necrotic enteritis. In the first experiment, cultures of C. perfringens were mixed with the feed at day 9, 10, 11, and 12, and the coccidial vaccine was given at day 10, whereas in the second experiment, C. perfringens cultures were mixed with the feed at day 17, 18, 19, and 20, and the coccidial vaccine was given at day 18. Chickens were examined at day 9, 11, 12, and 15 (Experiment 1), and at day 17, 18, 20, and 24 (Experiment 2). There was no mortality in any of the groups; however, chickens in the groups receiving both coccidial vaccine and C. peifringens developed the subclinical form of necrotic enteritis, demonstrated by focal necroses in the small intestine, whereas chickens in control groups or groups receiving only coccidial vaccine or only C. perfringens cultures developed no necroses. The results underline the importance of predisposing factors in the development of necrotic enteritis.